Elodie Czuba

Audrey Grunenberger

repair CPP Cell-penetrating peptide CRC Colorectal cancer CRISPR Clustered Regularly Interspaced

A big thank you to my colleagues at Divincell,

First and foremost, I would like to extend my heartfelt thanks to Virginie Faure and Gilles Divita, my thesis supervisors. Virginie, your constant support, guidance, and help have been invaluable to me. I am forever grateful for the time you dedicated to developing my soft skills. Gilles, I sincerely appreciate the trust you placed in me over these past years and the freedom I had in managing this project.

I would like to express my warmest thanks to Dr. Alice Carrier, Dr. Emmanuel Garcion, Dr. Jean-Luc Lenormand, and Dr. Nadir Bettache for agreeing to be part of my jury and for the time they dedicated to evaluating my work. I would also like to thank Dr. May Morris and Dr. Jessica Escoffier for their insightful comments and advice during the individual follow-up meetings for this project.

A special thank you goes to Dr. Jean-Luc Coll, the leader of our research team, for believing in me and giving me the opportunity to join this incredible team for my master internship and PhD research. I am deeply grateful to you for showing me the beauty of the French mountains and making me feel at home. It meant a lot to me. This project, to me, is a testament to the collaboration of kind-hearted individuals with whom I had the privilege to work. I would like to thank the entire team 5, including past and current members, for creating a positive atmosphere, providing advice, enjoying hikes, sharing coffee breaks, and Friday breakfasts, among many other things.

In particular, I would like to express my gratitude to Dr. Xavier Le Guével for your invaluable advice regarding the physico-chemical characterization and optimization of my nanoparticles. Specially, thank you for taking the time to discuss with me in my mother tongue and keeping me updated on Latin American events in the city. It always brightened my days.

I extend my thanks to Sean Moro, Pierre Le Lievre, Ghadir Kalot, Theo Ziegelmeyer, and Mohammed Omrani for sharing this journey with me. Thank you for encouraging breaks from work, engaging in unique discussions, and providing invaluable French lessons! I also want to thank Fabien Dalonneau for your motivation during the writing of this manuscript and for making me laugh even in the hardest moments. Special thanks to Camila Castillo and Nazareth Carigga for your support, delicious Latin food, and the beautiful friendships we have formed. It has been a true pleasure to work with each and every one of you on a daily basis.

I would like to express my warmest thanks to Tao Jia and Thibault Jacquet for training me in cell culture and nanoparticle characterization. Thank you for your encouragement to pursue a PhD in the nanomedicine field, it was greatly appreciated.

Table of content

List of abbreviations ________________________________________

Introduction _______________________________________________

1.

Deciphering the CRISPR-Cas9 system for precise genome editing 12 Genome editing is a technique that enables precise modifications to be made in the genome of a living cell, allowing scientists to change, remove or add specific genes. It can be performed in ex vivo culture and selection applied to enrich edited cells. It can also be performed antenatally in essentially any animal species. It has been used to edit singlecell embryos, which when implanted into surrogate mothers, develop into adults in which all cells are edited.

Genome editing has been studied since the 1970s and the first important achievement was when scientist demonstrated that they could integrate a DNA sequence into a host genome by homologous directed repair (HDR) when introducing a DNA containing homologous arms at both ends [START_REF] Capecchi | Altering the Genome by Homologous Recombination[END_REF]. This significant breakthrough marked the first time that desired changes could be introduced into the cell genome. However, using HDR alone brought many problems, such as low efficiency of integration of the external DNA and integration at random genomic locations. A significant progress in the field came when scientist were able to introduce well defined DNA double strand breaks (DSB) into the genome of eukaryotic cells [START_REF] Rouet | Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease[END_REF]. Moreover, scientists figured out that the introduction of an artificial DNA restriction enzyme into the cell induced the formation of double-stranded DNA (dsDNA) at specific recognition sites. These significant advances highlighted both the role of HDR and non-homologous end joining (NHEJ) repair mechanisms in gene editing and allowed for the first time to select a specific genomic target [START_REF] Cohen-Tannoudji | I-Sce I-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells[END_REF].

Mechanistic insights into site-specific genome editing

Genome editing using on site-directed nucleases is based on the cleavage of dsDNA by means of the nuclease followed by the activation of the cell's own DNA repair mechanisms: HDR or NHEJ. Four main mechanisms of site-specific genome editing have been developed: meganucleases (MegNs), zinc finger nucleases (ZFN), transcription activator like effector nuclease (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats associated protein Cas (CRISPR-Cas) (Figure 1). Once Cas9 finds a PAM site, if the gRNA binds to the DNA, a double break occurs three base pairs upstream the PAM) [START_REF] Premalatha | Genome Editing in Plant Biotechnology[END_REF].

Meganucleases (MegNs)

Meganucleases (MegNs) were the first endonuclease system used for gene editing. They are naturally produced by all forms of microorganisms and are found on eukaryotic mitochondria and chloroplasts as well. MegNs facilitate the lateral mobility of genetic elements within an organism, a process called homing, therefore, adopting the name of homing endonucleases. MegNs are characterized by having large recognition sites of approximately 14 to 40 base pairs in the target sequence (Figure 1A). This technique has the high specificity to the target DNA due to this long recognition site and generate low off target effects. It is easy to pack within a delivery vector due to its relatively small size and results in a high mutation rate due to the production of a 3' overhang after the endonuclease activity. However, some drawbacks related to MegNs are the difficulty finding an enzyme that targets the desired target locus and the low target recognition efficiency. Furthermore, this technique faces the engineering challenge of designing a sequence specific enzyme for each target sequence, which is time consuming and expensive [START_REF] Gaj | Genome-Editing Technologies: Principles and Applications[END_REF].

Zinc finger nucleases (ZFNs)

Zinc finger nucleases (ZFNs) are an engineered restriction enzyme used for genome editing. They consist of two components: a DNA cleaving endonuclease domain (often from the bacterial restriction enzyme called FokI) and a zinc finger domain that binds to a specific DNA sequence typically between 9 and 18 base pairs (Figure 1B). Each ZFN is typically composed of three to four zinc finger domains. The higher the zinc finger domains, the higher specificity and efficiency of the system. When the zinc finger domains and the DNA cleaving domain are bound together, they can specifically edit the gene by inducing DNA DSB. The major advantage of ZFNs over MegNs is that they are easier to design through the assembly of pre-existing zinc finger domains with known target sequences. Nevertheless, the major limitation of ZFNs is that its specificity relies not only on the target sequence, but also on the adjacent DNA sequences on the genome, called context-dependent specificity. This issue may induce not specific DNA cleavage that generates in turn genome fragmentation and instability.

TALE nucleases (TALENs)

The efforts to overcome the aforementioned limitations resulted in the development of new nucleases: transcription activator-like effector nucleases (TALENs). Like MegNs and ZFNs, TALENs require that the proteins are re-engineered for each targeted sequence. They are composed of an amino-terminal DNA binding domain derived from highly conserved repeats in transcription activator-like effectors fused to a carboxyl-terminal FokI cleavage domain (Figure 1C) [START_REF] Christian | Targeting DNA double-strand breaks with TAL effector nucleases[END_REF][START_REF] Miller | A TALE nuclease architecture for efficient genome editing[END_REF]. TALENs offer significant advantages over ZFNs and MegNs. First, there is no need of selection or directed evolution to engineer TALE arrays, reducing the time and complexity to assemble a new functional protein. Second, TALENs have improved specificity due to each module in the DNA binding domain recognizing just one nucleotide, compared by the three nucleotides recognized by each zinc finger domain. Avoiding the interference of recognition sequences allows the use of several modules and therefore increases the specificity. In addition, TALENs have shown higher efficiency to produce DSB and to reduce toxicity compared to ZFNs. Despite these significant improvements, the use of TALENs remains complex and it is confronted to delivery challenges due to the large size of the system.

CRISPR-Cas nucleases

The Clustered regularly interspaced short palindromic repeats associated protein Cas (CRISPR-Cas) is known as the third generation of genome editing tools. Unlike the previous generations where the interaction of the artificial protein with the DNA was required for recognition of the target sequence, CRISPR-Cas system recognizes the target sequence through RNA-DNA interactions (Figure 1D). Moreover, it should be emphasized that the use of CRISPR-Cas technology only needs the manipulation of a short RNA sequence and there is no need for complicated alterations in the protein domain. This system offers an easy design for any target DNA and the possibility of modifying several genomic sites simultaneously (multiplexing). CRISPR-Cas system represents a breakthrough in the field of genetic engineering and gene therapy. CRISPR-Cas, often compared to molecular scissors, enables precise and efficient modification of genomic DNA. Originally discovered as bacterial immune system, CRISPR-Cas systems have been adapted as a powerful and versatile gene editing tool used in a wide range of organisms, including plants, animals, and humans. At the same time, CRISPR-Cas has evolved as the simplest, less expensive editing tool [START_REF] Thurtle-Schmidt | Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates[END_REF].

During this project, we focused on the use of the CRISPR-Cas technology, which will be further discussed in the following sections.

Tracing the bacterial origins of CRISPR-Cas9

Prokaryotes organisms have suffered from numerous viral or parasitic infections. These viral attacks have pushed them to adapt and develop specialized mechanisms for innate and adaptive immunity defense. Although prokaryotes have a large repertoire of defense systems, by far the most widespread defenses are Restriction-Modification (RM) and CRISPR-Cas immune systems. Innate immunity most commonly relies on endonuclease cleavage of any incoming DNA that lacks a specific epigenetic modification, through the system known as RM. On the other hand, CRISPR-Cas adaptive immunity relies on the insertion of short DNA sequences from parasite genomes into CRISPR loci on the host genome to provide sequence-specific protection. Bacteria and archaea generate cellular memory of past viral invaders by recombining short viral DNA sequences into their genome. These sequences are then used to detect and destroy the multi-generational exposure (MGE) also called protospacers in subsequent infections.

The entry of foreign genetic material by viral infections or plasmid attack activate the CRISPR-Cas bacterial defense mechanism. The foreign material is recognized as non-self and three phases are activated: the adaptation (also called spacer acquisition), the expression and the interference, resulting in the degradation of the invader's DNA sequence [START_REF] Deveau | CRISPR/Cas system and its role in phage-bacteria interactions[END_REF][START_REF] Van Der Oost | CRISPR-based adaptive and heritable immunity in prokaryotes[END_REF].

First, during the adaptation step, short sequences homologous to the invading viral or plasmid DNA are integrated into the CRISPR locus of the host genome forming CRISPR arrays (Figure 2). The acquisition of these spacers is mediated by proteins that are highly conserved between different CRISPR systems, called Cas1 and Cas2. The selection of protospacers that have to be memorized depends on the presence of few nucleotides called PAM (Protospacer adjacent motive), which varies depending on the species of bacteria or archea (Table 1) [START_REF] Mojica | Short motif sequences determine the targets of the prokaryotic CRISPR defence system[END_REF].

Figure 2. Streptococcus' adaptive immune system against viruses: CRISPR-Cas9. Adapted from "2020 Nobel Prize in Chemistry: A Tool for Genome Editing (CRISPR-Cas9)", by BioRender.com (2020). Following infection, Cas1 and Cas2 mediate incorporation of short sequences of the viral genome as spacers within the bacterial CRISPR locus. At reinfection by the same virus, the CRISPR locus gets expressed as pre-crRNA, along with tracrRNA. The pre-crRNA is processed to yield guide RNAs (gRNAs) which bind the ribonucleoprotein Cas9 and target this complex to complementary sequences of the infiltrating bacteriophage genome, prompting its Cas9-mediated cleavage.

Table 1. Cas9 PAM sequences from different species. Adapted from [START_REF] Leonova | CRISPR/Cas9 technology in translational biomedicine[END_REF].

Second, after the acquisition of the spacers, CRISPR arrays are transcribed into CRISPR RNAs (pre-crRNAs) that are then processed to form the CRISPR machinery. There are several different CRISPR-Cas mechanisms that have been identified (Type I, II, III, IV, V and Type VI, see Figure 3). The most exploited system for genome editing is the Type II CRISPR-Cas system due to its simplicity (requiring only one Cas protein and two RNA components). Briefly, in type II CRISPR-Cas system, a second RNA molecule is transcribed from an upstream locus forming the trans-activating CRISPR RNA (tracrRNA). The tracrRNA binds to pre-crRNA forming a double strand RNA molecule that is cleaved by the RNAse III endonuclease. This results in the formation of crRNA:tracrRNA complexes that contain only one spacer sequence.

Finally, for the interference step, the system is ready to protect the host from a new attack of the invader's DNA. crRNA:tracrRNA complex binds to the Cas9 protein that searches the invading DNA for the 5'-NGG-3' PAM motif and verifies the complementarity with the crRNA. When Cas9 recognizes the specific PAM sequence located downstream of the cleavage site on the non-target DNA strand, it verifies the complementarity and then cleaves the DNA, resulting in protection from the invader.

To ensure that the system targets only the infectious DNA, and not the CRISPR array of the host genome, CRISPR-Cas effector complexes use the sequence flanking the complementary target for discrimination: in a CRISPR array, the flanking sequence consists of a CRISPR repeat sequence, whereas targets in an infectious genome are usually selected to be flanked by the conserved PAM sequence.

Cas9 ortholog PAM Reference Streptococcus pyogenes (SpCas9)

5′-NGG-3′ (N-any) [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF] Francisella novicida (FnCas9) 5′-YG-3′ (Y-Pirimidines) [START_REF] Hirano | Structure and Engineering of Francisella novicida Cas9[END_REF] Staphylococcus aureus (SaCas9) 5′-NNGRRT-3′ (R-Purines) (Xie et al., 2018) Streptococcus thermophiles (St1Cas9) 5′-NNAGAAW-3′ (W-A or T) [START_REF] Garneau | The CRISPR/cas bacterial immune system cleaves bacteriophage and plasmid DNA[END_REF] Streptococcus thermophiles (St3Cas9) 5′-NGGNG-3′ [START_REF] Magadán | Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system[END_REF]) Neisseria meningitidis (NmCas9)

5′-NNNNGATT-3′ (C. M. [START_REF] Lee | The neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells[END_REF] Neisseria meningitidis (Nme2Cas9) 5′-NNNNCC-3′ (Dayanand & Olivia E. Atherton1, Jennifer L. Tackett2, Emilio Ferrer1, 2018) Lactobacillus buchner (lbCas9)

5′-AAAA-3′ [START_REF] Briner | Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity[END_REF] Figure 3. Conventional classification of CRISPR-Cas systems [START_REF] Gostimskaya | CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing[END_REF].

Evolution of CRISPR-Cas9 in biotechnology: An overview

A summary of the history of the CRISPR-Cas9 system is shown on Figure 4. The history of this powerful technology begins with the identification of short DNA repetitive sequences of 24 nucleotides in E. Coli by Ishino et al. from Osaka University in 1987 [START_REF] Ishino | Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isoenzyme conversion in Escherichia coli, and identification of the gene product[END_REF]. At that time, sequencing of these difficult-to-study DNA fragments took several months and neither their origin nor their significance in the bacterial cell were understood by their discoverers. In 1993, a major advance was made when similar repeated sequences were identified by Mojica and co-workers in the archaeon Haloferax mediterranei during the research on regulatory mechanisms allowing extremely halophilic archaea to adapt to high salt environments [START_REF] Mojica | Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites[END_REF]. They hypothesized that these unusual loci include fragments of foreign DNA and are, in fact, a part of the immune system of bacteria and archaea.

The invention of the automated sequencing machines and development of efficient procedures for DNA sequencing during the 90s provided scientists for the first time with access to complete genome sequences. The structural and functional characterization of the CRISPR-Cas system started in 1993 when numerous regularly spaced repeated sequences were identified in more models of bacteria and archae, increasing their biological relevance [START_REF] Jore | RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements[END_REF]. The repeated DNA sequences were entitled as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) by Ruud Jansen and collaborators in 2002 [START_REF] Jansen | Identification of genes that are associated with DNA repeats in prokaryotes[END_REF]. Nearby genes were found to be accompanying the CRISPR genes and were, therefore, called CRISPR-associated (Cas) genes. From 2005 to 2011, the bacteriophage origin and the link between the functions of CRISPR and Cas genes were analyzed, resulting in its identification as part of the prokaryotic immune system [START_REF] Barrangou | CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes[END_REF][START_REF] Jore | RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements[END_REF][START_REF] Makarova | A putative RNA-interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action[END_REF][START_REF] Makarova | Evolution and classification of the CRISPR-Cas systems Kira[END_REF][START_REF] Mojica | Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[END_REF][START_REF] Pourcel | CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies[END_REF].

Since 2011, the application phase of CRISPR-Cas systems is ongoing. In 2012, Jinek et al. laid the foundation to what would ultimately become a revolution in genome editing and transcriptional control. They mentioned that in Type II systems, the dual guide RNA complex tracrRNA:crRNA of Cas9 could be fused into a single chimeric RNA by linking the 3′ end of crRNA to the 5′ end of tracrRNA [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF]. Such a technique would allow for programmed DNA cleavage through engineering of the chimeric RNA molecule, later designated as sgRNA (single guide RNA). Shortly thereafter, further discoveries unraveled the full potential of CRISPR as a tool for genetic editing.

In 2013, Jiang et al. used the CRISPR-Cas9 system to induce targeted mutations in the genome of Streptococcus pneumoniae and E. coli strains (Wenyan Jiang, David [START_REF] Jiang | 5′-NNGRRT-3′ PAM for Sufficient Cleavage and Possesses[END_REF]. During the same year, Bikard et al. demonstrated how CRISPR could be used as a new tool to regulate gene expression by either activating or repressing the transcription of bacterial genes [START_REF] Bikard | Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system[END_REF]. Then, CRISPRmediated manipulation was tested on human cell cultures, mice, plants, and yeasts. The list of tested models continues increasing.

The first human clinical studies using CRISPR-Cas9 commenced in October 2016 in China [START_REF] Lu | Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer[END_REF]. The PD-1 (Programmed Cell Death 1) gene was inactivated ex vivo in blood cells in the hope that such modified cells, would attack the non-small-cell lung cancer that the patient suffered from when returned to circulation. They concluded that clinical application of CRISPR-Cas9 gene-edited T cells was generally safe and feasible and that future trials were required to improve therapeutic efficacy. In the U.S., ex vivo therapy using CRISPR-Cas9 was first performed in July 2019 on a patient with sickle cell anemia (CRISPR Therapeutics, founded by Charpentier). The therapy significantly improved the patient's condition for at least a few months after the procedure.

The employment of CRISPR-Cas systems has grown every year to thousands of research around the world [START_REF] Gostimskaya | CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing[END_REF]. Dr Emmanuelle Charpentier and Dr Jennifer A. Doudna received the 2020 Nobel Prize in Chemistry for their key insight contributions to the development of the groundbreaking new technology CRISPR-Cas in 2020.

The discovery of CRISPR-Cas9 as an immune system in prokaryotes at the turn of the 20th-21st centuries has led to a revolution in the field of genomic manipulations. This ground-breaking technique is a worthy continuation of such exciting scientific events as the publication of the double-stranded structure of DNA by Watson and Crick in 1953, the birth of the first child by in vitro fertilization in 1978, and the cloning of Dolly the sheep in 1996.

Decoding CRISPR-Cas9: Components for precise gene editing

Type II CRISPR systems are the simplest and most exploited ones for gene editing. It requires only the presence of one nuclease, such as Cas9, and two RNA components (crRNA and tracrRNA). In 2012, J. Doudna and E. Charpentier greatly facilitated the use of CRISPR-Cas9 as genetic scissors. The researchers used their knowledge about crRNA and tracrRNA to fuse the two into a single molecule, which they called single guide RNA (sgRNA) [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF].

The sgRNA is an engineered RNA molecule that serves as a guide for the CRISPR-Cas9 system to find and target a specific DNA sequence. It is composed of two parts: at the 3' end, a 100 nucleotides sequence forming a tertiary double-stranded structure that allows the binding of the Cas9 protein; and at the 5' end, a 20 nucleotides sequence that serves as a model for the Cas9 search of complementary DNA sequence. sgRNA is designed to be complementary with any DNA of interest, the only requirement for the target DNA is to be adjacent to a PAM sequence (3 to 5 nucleotides). There are now several computational tools and software that facilitate the sgRNA design [START_REF] Cui | Review of CRISPR/Cas9 sgRNA Design Tools[END_REF].

The other component of the system is the Cas9 protein from S. pyogenes (SpCas9), which consists of 1368 amino acids and is a multidomain and multifunctional endonuclease. Structural analysis of SpCas9 revealed its composition by 5 domains. A first region called recognition (REC) lobe, formed by REC and BH domains, is responsible for sgRNA binding and initiating cleavage activity. The REC lobe plays a major role on Cas9 specificity by controlling the cleavage of off-target sequences [START_REF] Palermo | Key role of the REC lobe during CRISPR-Cas9 activation by "sensing", "regulating", and "locking" the catalytic HNH domain[END_REF]. The PI (PAM interacting) domain helps to stabilize the sgRNA and Cas9 complex, positions the Cas9 protein relative to the PAM sequence and contributes to the local DNA strand separation.

The HNH domain cuts the target DNA strand complementary to the sgRNA while the RuvC domain nicks the strand that is not complementary to the sgRNA (Figure 5A).

After conformational rearrangement, Cas9 protein binds to the sgRNA and pass into an active state (Figure 5B). Once it is activated, Cas9 protein can bind to the DNA molecule to search for a PAM motive and a complementary region to the sgRNA (Figure 5C). Finally, if the DNA meets the complementarity criteria, Cas9 activates the nuclease domains, generating blunt ends 3 to 5 nucleotides upstream of the PAM region (Figure 5D).

Navigating DNA double-strand break repair mechanisms

The gene editing capacity of the CRISPR-Cas9 system relies on the DNA repair mechanisms proper of the target cell. DNA Double Strand Break (DSB) caused by Cas9 endonuclease is among the most genotoxic DNA damage. If left unrepaired, they can induce genomic instability, chromosomal rearrangements and cell death [START_REF] Frank | DNA repair pathway choices in CRISPR-Cas9 mediated genome editing[END_REF]. Cells have evolved to repair this damaged DNA through different pathways. The two major mechanisms are: the error-prone nonhomologous end joining (NHEJ) repair and the error-free homology-directed repair (HDR) (Figure 6). Mammalians cells activate the DNA damage response (DDR) by delaying the cell cycle progression in order to avoid the transmission of incorrect genetic information and activating control mechanisms. First, early sensor proteins are rapidly recruited to the DNA DSB site (Figure 7). The Mre11-Rad50-Nbs1 (MRN) complex recognizes and binds to the broken ends of the DNA and processes them by recruiting and activating the kinase Ataxia Telangectasia Mutated (ATM) [START_REF] Falck | Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage[END_REF]. Activated ATM triggers the phosphorylation of the histone variant H2A.X, facilitating the accumulation of repair and checkpoint proteins in a large adjacent region to the DNA break site [START_REF] Kinner | c -H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin[END_REF].

Other proteins are recruited to the break site, including the Breast Cancer 1, early onset (BRCA1), and p53-binding protein 1 (53BP1) (J. [START_REF] Lee | 53BP1 promotes ATM activity through direct interactions with the MRN complex[END_REF][START_REF] Yun | Understanding the functions of BRCA1 in the DNA-damage response[END_REF]. The DNA repair mechanism is chosen depending on the accumulation of BRCA1 and 53BP1. BRCA1 mediates an homologous directed repair mechanism while 53PB1 mediates a non-homologous end joining repair.

Figure 7. Early steps of DNA damage response. DNA DSB is detected by MRN complex that recruits DNA damage sensing proteins to the break site. The accumulation of these proteins lead to the initiation of HDR or NHEJ DNA repair mechanisms [START_REF] Brochier | Chromatin Modifications Associated with DNA Double-strand Breaks Repair as Potential Targets for Neurological Diseases[END_REF].

The homology directed repair HDR is a high fidelity, error-free DNA repair mechanism. It requires a DNA template for the DNA DSB repair. HDR relies on a vast processing of the DNA at the break site mediated by MRN and its interacting partner CtIP (C-terminal binding protein interacting protein) (Figure 8). A 3' single stranded DNA is created where Rad51 assembles to generate a ribonucleoprotein filament and to promote the search for an homologous template. The DNA strand invasion and consecutive recombination is facilitated by Rad52,Rad54,and multiple Rad51 paralogs (Xrcc2,Xrcc3,Rad51L2,Rad51L3). HDR can only be used by dividing cells, when the sister chromatids are available to be used as DNA repair template or with an external DNA provided for gene editing.

In contrast, the classical non-homologous end joining repair (cNHEJ) mechanism is used by most cells and does not require an undamaged template DNA. It involves the direct ligation of the broken ends with minimal DNA processing. Specialized proteins recognize, resect, polymerize and ligate the DNA ends [START_REF] Frank | Non-homologous DNA end joining and alternative pathways to doublestrand break repair[END_REF]. Importantly, this mechanism is error prone, meaning that the resulting DNA junctions often contain DNA deletions or insertions (<10 nucleotides). cNHEJ initiates with the binding of the ring-shaped protein heterodimer Ku70/Ku80 (also known as XRCC6-XRCC5) to each end of the DNA DSB (Figure 8). Ku70/Ku80 protect the broken strands from further degradation and recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs). If the DNA ends are not suitable for direct ligation, the nucleases Artemis or Exo1 process the damaged or mismatched nucleotides and the DNA polymerases Polλ and Polμ fill the gap [START_REF] Daley | DNA joint dependence of Pol X family polymerase action in nonhomologous end joining[END_REF][START_REF] Niewolik | DNA-PKcs dependence of artemis endonucleolytic activity, differences between hairpins and 5′ or 3′ overhangs[END_REF]. After the DNA ends are made compatible, DNA-PKcs recruits the DNA ligase IV (LIG4), the associated scaffolding factors XRCC4, XRCC4-like factor (XLF), paralogue of XRCC4 and XLF (PAXX) for the DNA ligation [START_REF] Mcelhinny | Ku Recruits the XRCC4-Ligase IV Complex to DNA Ends[END_REF]. Additional DNA repair mechanisms are the microhomology mediated end joining (MMEJ), also known as alternative end joining (alt-EJ), and the single-strand annealing method (SSA), both of which are error-prone [START_REF] Frank | DNA repair pathway choices in CRISPR-Cas9 mediated genome editing[END_REF]. As cNHEJ, MMEJ and SSA do not require a DNA template to repair the DSB [START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway[END_REF]. Instead, MMEJ use short (5-25 base pairs) microhomologous sequences present near the broken DNA ends to realigned them [START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway[END_REF]. Similarly, SSA require a 3′ ssDNA overhangs for the annealing of homologous repeats [START_REF] Bhargava | Regulation of Single-Strand Annealing and its Role in Genome Maintenance[END_REF].

After Cas9-induced DSBs, Cas9 asymmetrically releases the DNA. The PAM-proximal end of the cleaved DNA is released within minutes while the PAM-distal end of the DSB remains bound by Cas9 for longer time [START_REF] Liu | Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition[END_REF]. As a result, the PAM-proximal DSB end is exposed for repair, while the PAM-distal end may be temporally blocked by the still bound Cas9 [START_REF] Clarke | Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks[END_REF]. RNA-polymerase and the histone chaperone FACT (FAcilitates Chromatin Transcription) can remove Cas9 from the PAM-distal side. However, the mechanisms behind this process during gene editing and how the asymmetrical release of Cas9 affects the DSB repair remain unclear.

Factors influencing DNA repair outcomes of Cas9-induced doublestrand breaks

The DNA repair outcomes following Cas9-induced double-stranded breaks (DSBs) can vary depending on a variety of factors, including the cell type being edited, the stage of the cell cycle, the target site sequence and chromatin structure and the DNA donor template for HDR mediated repair. Understanding the factors that influence DNA repair outcomes is critical for optimizing gene editing protocols and achieving the desired genetic modifications.

Cell cycle

The choice of the DNA repair pathway used by the cell highly depends on the cell cycle stage of the cell. The NHEJ repair is predominant during G1, S and G2 phases, while the HDR is limited to the end of the S phase and G2 phase (Figure 9). The arrest of the cell cycle on S/G2 phase using aphidicolin promotes the repair of Cas9-induced DSB through HDR, MMEJ and SSA [START_REF] Lin | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery[END_REF]. Similarly, fusing Cas9 to the N-terminal region of the protein Geminin, leads to its proteolytic degradation during G1; therefore, restricting Cas9 activity to S/G2 phases and enhancing HDR repair mechanism [START_REF] Gutschner | Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair[END_REF]. During G1 phase, NHEJ dominates by more than 50-fold for the repair of DSB. Even during S phase and G2 phase, the ratio of NHEJ to HDR is estimated to be 4:1 [START_REF] Beucher | ATM and Artemis promote homologous recombination of radiation-induced DNA doublestrand breaks in G2[END_REF]. Target site sequence and chromatin structure DNA repair outcomes after Cas9-induced DSB differ substantially depending on the location within the genome [START_REF] Chakrabarti | Target-Specific Precision of CRISPR-Mediated Genome Editing[END_REF]. However, DNA editing results are not completely random [START_REF] Shen | Predictable and precise template-free CRISPR editing of pathogenic variants[END_REF]. Similar repair events were observed when a single protospacer targeted various genomic sites [START_REF] Van Overbeek | DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks[END_REF]. Research indicates that editing outcomes can also be influenced by nucleotides surrounding the Cas9 cleavage site [START_REF] Allen | Predicting the mutations generated by repair of Cas9-induced double-strand breaks[END_REF][START_REF] Chakrabarti | Target-Specific Precision of CRISPR-Mediated Genome Editing[END_REF][START_REF] Shen | Predictable and precise template-free CRISPR editing of pathogenic variants[END_REF].

Additionally, Cas9 cleavage efficiency is influenced by the accessibility of the DNA. Nucleosome breathing and alterations in nucleosome architecture caused by chromatin remodeling proteins can enhance Cas9 cleavage efficiency [START_REF] Isaac | Nucleosome breathing and remodeling constrain CRISPR-Cas9 function[END_REF]. The selection of DSB repair pathways is also influenced by the nearby chromatin structure. Heterochromatin and euchromatin have varying effects on DNA repair [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF]. Heterochromatin, characterized by H3K9 trimethylation marks (H3K9me2/3), supports HR, MMEJ, and SSA, while cNHEJ is favored in euchromatin [START_REF] Schep | Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance[END_REF]. The precise mechanisms by which local chromatin structure affects DNA repair pathways remain uncertain, and ongoing studies aim to uncover how chromatin structure impacts Cas9-mediated genome editing.

Homology directed repair with ssDNA or dsDNA donor templates

In CRISPR-Cas9 induced-DSB repair, template-dependent DSB repair pathways rely on an exogenous donor template for precise gene editing. The double-stranded DNA donor templated repair (DSTR) primarily occurs through the HDR pathway, while the singlestranded DNA donor templated repair (SSTR) mainly takes place through SSA.

Hence, efforts are being made to regulate the DSB repair pathways utilized by cells during genome editing. It's worth noting that the HDR process is considerably less efficient than NHEJ, and gene knock-in or replacement via CRISPR-Cas9 has lower efficacy than gene knock-out. However, the mechanisms governing DSB repair are extremely intricate and still not entirely understood.

Challenges associated to CRISPR-Cas9 delivery and gene editing

CRISPR-Cas9 has shown great potential as a gene editing system. Nonetheless, there are still some challenges that need to be addressed (Figure 10):

Encapsulation:

The big size of Cas9 plasmid, mRNA or protein make it difficult to pack the system into a single vector. For instance, Cas9 protein is bigger than most proteins ( 160 kDa) and is positively charged. Most vectors are designed to be positively charged, so they cannot encapsulate Cas9 protein through electrostatic interactions. Cas9 mRNA and plasmid are larger than most cargo molecules as well, making it difficult to develop suitable vectors. All these defects need to be taken into consideration in future non-viral vector designing.

Stability:

It is important to guarantee the stability of the system when it is in physiological condition. Cas9 plasmid, mRNA or protein can be easily degraded by nucleases and proteases present in the blood. The system might also interact with plasma proteins that facilitate its recognition by the reticulo endothelial system (RES), clearing and eliminating the system before its gene editing activity. PEGylation is a widely employed approach to mitigate opsonization and reduce clearance by the RES and it has shown promise in vector-mediated CRISPR/Cas9 applications. Nevertheless, it is important to consider the potential drawback of decreased cellular uptake associated with PEGylation. Therefore, the appropriate delivery systems to guarantee stability of CRISPR-Cas9 are required.

Immune response and cytotoxicity:

Due to the bacterial origin of the Cas9 proteins, when introduced directly to the body, the CRSIPR-Cas9 system could induce an immunological response [START_REF] Crudele | Cas9 immunity creates challenges for CRISPR gene editing therapies[END_REF]. SpCas9, originating from Streptococcus pyogenes and SaCas9 derived from

Staphylococcus aureus, elicit a significant immune response. This reactivity is attributed to the frequent invasion of these bacteria into the human body, resulting in diseases. Indeed, pre-existing anti-Cas9 IgG antibodies against spCas9 and SaCas9 were found in 65% and 79% of healthy human samples, respectively diseases [START_REF] Charlesworth | Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans Carsten[END_REF][START_REF] Colque-Navarro | Levels of antibody against 11 Staphylococcus aureus antigens in a healthy population[END_REF]. Moreover, Charlesworth et al demonstrated the presence of antigen specific T-cells against SaCas9 in 46% of the donor samples. Even though specific T-cells against spCas9 were not detected, the absence of such cells could not be concluded due to low sensitivity levels of the assay [START_REF] Charlesworth | Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans Carsten[END_REF]. These studies demonstrated that the humoral and adaptive immune response to Cas9 proteins should be carefully considered for CRISPR-Cas9 gene editing studies. By enclosing these components within the internal compartments of the vectors, the process of recognition and subsequent elimination would be substantially hindered.

It was also reported that the overexpression of Cas9 protein for long time periods (< 3 days) in mammalian cells generates significant toxicity [START_REF] Yu | The Cytotoxic Effect of RNA-Guided Endonuclease Cas9 on Human Hematopoietic Stem and Progenitor Cells (HSPCs)[END_REF]. The transient transfection of Cas9 mRNA and modifications of Cas9 to shorten its half-life are strategies that have been used to diminish possible cytotoxic effects [START_REF] Frank | Non-homologous DNA end joining and alternative pathways to doublestrand break repair[END_REF].

Off target mutations:

At the molecular level, additional factors need to be addressed to minimize CRISPR-Cas9 possible off-target effects. The presence of multiple DNA sequences with high similarity with the target DNA favors off-target cuttings of the system. The optimization of the sgRNA to have minimal mismatches with the target DNA sequence is required to decrease possible off-target activity. Moreover, long exposure times to Cas9-sgRNA complex increases the risks of unintended cuts in the genome of the targeted cells.

Targeted delivery:

One major challenge is to ensure that the CRISPR-Cas9 components reach the correct target cells, especially in complex tissues and organs, where specific cell types are distributed in a heterogenous manner. To boost cellular uptake of the system, targeting ligands, such as antibodies, small molecules or short peptides can be introduced [START_REF] Torchilin | Tatp-mediated intracellular delivery of pharmaceutical nanocarriers[END_REF]. However, this modification could bring additional difficulties in incorporating the new molecules into the vector. Therefore, the successful CRISPR-Cas9 gene editing activity strongly depends on the efficiency of the chosen delivery system.

The selection and design of a safe and efficient delivery strategy for CRISPR-Cas9 remain the major obstacle for its use in vivo. A synopsis of the existing delivery strategies will be summarized on section 1.8 while potential future delivery candidates based on nanocarriers will be discussed on section 2.

Ethical issues:

The CRISPR-Cas9 system offers great potential for curing life-threatening illnesses, but its use raises several ethical and technical issues. These concerns are amplified when considering genetic modifications that can be passed down through generations [START_REF] Shinwari | Ethical issues regarding crispr-mediated genome editing[END_REF]. The permanent editing of the human genome to remove diseasecausing mutations or enhance desirable traits through beneficial gene insertion is a subject of ongoing debate. Non-reproductive cell genome modifications are not inheritable, while germ cell modifications can be passed on to future generations, which has led to concerns about the safety and dignity of human germline modification using CRISPR/Cas9-based gene editing [START_REF] Bailey | CRISPR-Mediated Gene Editing: Scientific and Ethical Issues[END_REF]. There are also worries about the potential for genocide. As a result, efforts have been made to stop the progress of human genome research until a global agreement is reached on society's acceptance of this new technology.

In biomedicine, moral concepts are based on empirical research and involve assessing potential risk-benefit ratios to maximize benefits while minimizing risks. When making moral decisions, it's crucial to consider the range of possible outcomes, their likelihood, and the various arguments for each outcome. CRISPR genome engineering technology raises at least three major ethical concerns. The first concern is related to the power and technical limitations of CRISPR, such as low on-target editing efficiency [START_REF] Peng | Potential pitfalls of CRISPR/Cas9-mediated genome editing[END_REF], incomplete editing (mosaicism) [START_REF] Tu | Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos[END_REF], and inaccurate on-or off-target editing [START_REF] Zischewski | Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases[END_REF]. However, the technology is evolving rapidly to overcome these limitations. The second concern refers to the long-term survival of modified species, including the potential for indefinite influence and the inheritance of edited genes, which could lead to unexpected consequences for future generations. It's important to address these ethical concerns and weigh them against the potential benefits of CRISPR technology before using it.

In France, as in many European countries, the use of CRISPR-Cas9 gene editing technology in humans is subject to strict regulations [START_REF] Hirano | Structure and Engineering of Francisella novicida Cas9[END_REF]. In 2018, the French Parliament passed a bioethics law that allows gene editing for research purposes but prohibits its use for producing genetically modified embryos for reproductive purposes [START_REF] Boggio | Human germline genome modification and the right to science: A comparative study of national laws and policies. Human Germline Genome Modification and the Right to Science: A Comparative Study of National Laws and Policies[END_REF].

Despite the potential ethical and safety concerns, the CRISPR-Cas9 technology has the ability to greatly improve health and wellbeing if utilized correctly. One of the main reasons to allow the use of this technology is to protect patients, particularly in instances where germline editing research is implemented in a clinical setting to prevent the inheritance of a specific genetic condition. This can alleviate the emotional burden that parents may face when they consider the possibility of their child being born with a genetic disease. Recent advancements, such as the modification of the Cas9 enzyme to limit mutations outside of its target region [START_REF] Ikeda | High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes[END_REF][START_REF] Ledford | Enzyme tweak boosts precision of CRISPR genome edits[END_REF], have improved the safety profile of CRISPR/Cas9 . Furthermore, the error rate of this technology can potentially be further reduced with additional modifications. It is important to note that the risk profile of CRISPR/Cas9 varies based on the design and ethical considerations, and careful usage is crucial for its success in improving health and quality of life.

Figure 10. Challenges and overcoming strategies of CRISPR/Cas9. Immunogenicity, off-targeting, polymorphism, delivery technique, and ethical issues are main limitations, difficulties and challenges of the CRISPR/Cas9 system in clinical trials and its recent discovery and usage in humans [START_REF] Rasul | Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy[END_REF].

Exploring diverse CRISPR-Cas9 delivery approaches

The CRISPR-Cas9 gene editing system can be delivered in three different modes: as plasmid DNA, mRNA and ribonucleoprotein complex (RNP) (Figure 11).

Figure 11. CRISPR/Cas9 system delivered using pDNA, mRNA or RNP. The pDNA form follows a pathway where that converges into the mRNA pathway, and the mRNA form follows a pathway that converges into the RNP pathway. In the pDNA pathway, successful delivery of the plasmid leads to its transportation to the nucleus, enabling transcription to take place. The transcribed sgRNA and Cas9 mRNA are then exported from the nucleus to the cytoplasm. In the mRNA pathway, the mRNA molecule is translated, giving rise to the folded Cas9 endonuclease, which subsequently interacts with the sgRNA to form the Cas9 RNP complex. Finally, in the Cas9 RNP pathway, the RNP complex is imported into the nucleus, where the process of genome editing occurs [START_REF] Sinclair | Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing[END_REF].

Each CRISPR-Cas9 delivery mode has its own advantages and challenges to overcome, yet all of them have general gene editing effectiveness (Table 2). The first approach consists in the encoding of Cas9 protein and its sgRNA into a DNA plasmid. At the laboratory level, this delivery mode provides numerous advantages such as low cost, simple operation, and higher stability than the mRNA or RNP modes. How ever the big size of the Cas9 gene (4 to 7 Kb) and total plasmid size (9.3 Kb) pose great difficulties for the delivery methods [START_REF] Ran | In vivo genome editing using Staphylococcus aureus Cas9[END_REF]. CRISPR-Cas9 delivered in its DNA form must cross, in addition to the cell membrane, the nuclear barrier. DNA must be transcribed in the nucleus to its mRNA form before been translated into the Cas9 protein, delaying the onset of the system. DNA-encoded CRISPR-Cas9 shows lower gene editing efficiency with delayed activity during which the Cas9 protein remain active for longer time, increasing possible off target effects and cytotoxic activity (Mhatre V. Ho, Ji-Ann [START_REF] Mhatre | Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression[END_REF]. Moreover, delivering plasmid DNA encoding for CRISPR-Cas9 with viral vectors substantially raises the risk of permanent recombination in the genome [START_REF] Hanlon | High levels of AAV vector integration into CRISPR-induced DNA breaks[END_REF]. Nevertheless, plasmid delivery remains the most implemented delivery mode for CRISPR-Cas9.

CRISPR-Cas9 delivery modes

The second delivery mode for CRISPR-Cas9 system implies the RNA delivery of its components. As no transcription is needed, delivered Cas9 mRNA and sgRNA induced faster editing of the targeted gene than the plasmid approach. To have a functional assembly of the CRISPR-Cas9 machinery, the Cas9 protein and the sgRNA have to be available at the same time. To guarantee this, researchers have introduced the sgRNA encoded with the viral genome and Cas9 mRNA, so that the sgRNA is transcribed in the time that the mRNA is translated into the Cas9 protein. However this strategy implies the additional nuclear barrier that might reduce the efficiency of the system (C. [START_REF] Jiang | A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo[END_REF]. Another strategy consisted in the delivery of the Cas9 mRNA alone, followed by the delivery of the sgRNA 6 hours later [START_REF] Yin | Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo[END_REF]. This approach might synchronize the availabilities of Cas9 and the sgRNA but could also induce double stress to the cells due to the two transfections, depending on the delivery strategy used.

Moreover, the delivery of the system in its RNA form allows the transient expression of the Cas9 protein. This favors the control of the dose and exposure time to the Cas9 nuclease, limiting the risks of off target effects or cytotoxicity. However, the shorter availability of the system might reduce the gene editing efficiency. The major challenge for this approach is the poor stability due to the sensitivity of RNA molecules to enzyme degradation. It requires the design of an appropriate delivery system that protects the RNA cargo from RNAse degradation.

The third approach is to directly deliver the Cas9 protein and its sgRNA. It provides the fastest gene editing because no transcription or translation of the components are needed. As the RNA approach, it provides a transient expression of Cas9 protein that reduces the risk of off target effects or cytotoxicity. Some reports have successfully used this approach to generate efficient gene editing [START_REF] Park | Dna-free genome editing via ribonucleoprotein (RNP) delivery of CRISPR/Cas in lettuce[END_REF][START_REF] Wang | Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles[END_REF].

However, the big size of the Cas9 protein ( 160 kDa) and the sgRNA ( 31 kDa) make it difficult to pack into a delivery vector. The sudden introduction of big doses of a foreign protein into mammalian cells could lead to immune system response to eliminate the gene-edited cells. In addition, a highly pure Cas9 protein must be used to guarantee no bacterial endotoxin contamination. Often the Cas9 protein and sgRNA are synthesized in vitro and administered as Cas9/sgRNA RNP. The Cas9 protein production process is more difficult than obtaining pure plasmid or mRNA molecules and is certainly expensive.

Different strategies have been proven for the delivery of the CRISPR-Cas9 system for gene editing. They can be classified in three general groups: physical methods, viral methods, and non-viral methods (Figure 12). Adapted from [START_REF] Taha | Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges[END_REF].

➢ Physical delivery methods

Physical delivery methods temporally disrupt the physical barriers of the cells and do not require the intervention of delivery vehicles. The main representative techniques for this strategy are: electroporation, sonoporation and microinjection.

Electroporation:

Electroporation is a widely used technique to introduce proteins and nucleic acids into cells in vitro. It applies an electrical field to create nanometer-sized pores on the cell membrane and temporarily increase its permeability. Cells are first exposed to a solution

Physical methods

Viral methods

Non-viral methods containing CRISPR-Cas9 components and then they are subjected to high voltage electrical currents, creating pores that allow the diffusion of the components into the cell. Electroporation has several advantages as CRISPR-Cas9 delivery method. It can be used in a wide range of cell types, including primary cells and stem cells, and is particularly effective for cells that are difficult to transfect with traditional methods. Electroporation is suitable for any CRISPR-Cas9 delivery mode (Plasmid, combination of mRNA and sgRNA or ribonucleoprotein complex). Electroporation have been reported to yield high gene editing efficiencies using plasmid based CRISPR-Cas9 delivery mode to spinal cord cells, cancer cells, and embryonic cells. [START_REF] Fei | Tissue-and timedirected electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration[END_REF][START_REF] Hou | Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis[END_REF][START_REF] Mandal | Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9[END_REF][START_REF] Yang | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering[END_REF]. The RNP complex has been delivered through electrophoresis to mice zygotes, impacting the mice coat color efficiently (Figure 13)(S. [START_REF] Chen | Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes[END_REF]. High RNP delivery yields were reached on fibroblast, embryonic stem cells and T cells as well (S. [START_REF] Kim | Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins[END_REF][START_REF] Schumann | Generation of knock-in primary human T cells using Cas9 ribonucleoproteins[END_REF]. Similarly, mRNA and sgRNA CRISPR-Cas9 mode was introduced through electroporation into mice zygotes to generate a new mouse model [START_REF] Qin | Efficient CRISPR/cas9-mediated genome editing in mice by zygote electroporation of nuclease[END_REF].

However, electroporation has low targeting capability and could induce irreversible damage to the cells that lead to significant cell death. In addition, the requirement of special equipment difficult its use for in vivo applications.

Figure 13. Electroporation delivery of CRISPR-Cas9 RNP components. Targeting the Tyr gene impacts mice coat color. Fertilized embryos were combined with pre-assembled Cas9/sgRNA RNPs for electroporation and then transferred to pseudopregnant mothers to generate CRISPR edited mice. RFLP genotyping was made to validate Tyr gene editing. Bar plot quantifies coat color phenotypes generated after electroporation at 1msec length pulse and 3ms length pulse (S. [START_REF] Chen | Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes[END_REF].

Microinjection:

Delivering CRISR-Cas9 components by microinjection method has become a common laboratory technique as it reaches efficiencies approaching 100% [START_REF] Horii | Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering[END_REF][START_REF] Yang | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering[END_REF]. This method applies mechanical force to the cell membrane using a micro-scale needle to pierce the cell membrane and deliver the cargo directly to the target site within the cell. Microinjection can be used to deliver the plasmid encoding for Cas9 and sgRNA directly to the nucleus [START_REF] Chuang | Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors[END_REF][START_REF] Nakagawa | Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes[END_REF]. However, this strategy risks of random integration of single stranded DNA, and even circularized plasmids, into the host genome, creating constitutive expression of Cas9 that could produce more off target effects (H. [START_REF] Yang | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering[END_REF].

Ideally, the delivery of Cas9 mRNA and sgRNA would be in the cytoplasm and directly into the nucleus, respectively. Nevertheless, the technical difficulty and laborious process to do two different microinjections into a single cell make this option impractical. In addition, two microinjections in a single cell, even spaced by several hours, induce cell death (H. [START_REF] Yang | One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering[END_REF]. Therefore, the delivery of the RNA components of CRISPR-Cas9 is often done to the cell cytoplasm, where mRNA is translated into the Cas9 protein that complexes with the sgRNA and carries it to the nucleus. Most studies using microinjection method apply this delivery mode. For instance, it has been used to simultaneously knock-out four different genes in rat zygotes [START_REF] Ma | Genome modification by CRISPR/Cas9[END_REF] and to disrupt two different genes in monkeys embryos (Figure 14) [START_REF] Niu | Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[END_REF]. Less frequently but equally possible, microinjection is used to deliver the CRISPR-Cas9 RNP complex directly to the nucleus [START_REF] Nakagawa | Ultrasuperovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice[END_REF]. Microinjection is not limited by the size of the cargo molecules which is a significant limiting factor for viral vector delivery systems. It provides controllable dosage and precise delivery of CRISPR-Cas9 system into single cells, improving control over possible off target effects. Nevertheless, this technique requires professional microoperating equipments and high labor intensiveness. As only a single cell can be targeted for each microinjection, this technique is only suitable for a limited number of cells and has low throughput.

Figure 14. Physical delivery of CRISPR-Cas9 by microinjection method in cynomolgus monkeys. After fertilization of monkey oocytes by intracytoplasmic sperm injection, Cas9 mRNA and sgRNAs targeting Ppar-γ and Rag1 were co-injected into one-cell-stage embryos. Ppar-γ gene is involved in the regulation of adipocyte differentiation and Rag1 gene is involved in the generation of diverse antigen receptors in lymphocytes. Injected zygotes were then transferred into surrogate females and a set of twin female babies (A and B) were successfully delivered at full term. Simultaneous disruption of two target genes in A and B monkeys was confirmed by T7EN1 cleavage assay. Con represents a control wild-type Cynomolgus monkey (Con) [START_REF] Niu | Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[END_REF].

Sonoporation:

Sonoporation or acustoporation is a method that generate transcient permeabilization of the cell membrane using ultra sound waves (Figure 15A) [START_REF] Fajrial | A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing[END_REF]. The acustic waves disrupt the integrity of the cell membrane generating transient pores that allow the entry of the CRISPR-Cas9 components. Microbubbles could be added to the system to improve cell membrane perforation efficiency. The bubbles expand or shrink depending on the pressure and cause shock waves when they burst. For instance, sonoporation was used to insert the RFP (Red Fluorescence Protein) and GFP (Green Fluorescence Protein) genes after CRISPR-Cas9 HDR in Hela cells (Figure 15A) [START_REF] Yoon | Acoustic-transfection for genomic manipulation of singlecells using high frequency ultrasound[END_REF]. Plasmid encoding for Cas9 and sgRNA have been acustotransfected into human endometrial (HEC)-1A cancer cells to knock-out the epidermal growth factor receptor 2 (C-erbB-2) gene associated with breast cancer [START_REF] Cai | Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells[END_REF]. Sonoporation has also been applied to target the steroid type II 5-alpha-reductase (SRD5A2) gene which is involved in male pattern baldness [START_REF] Ryu | Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy[END_REF].

Compared to other physical methods, sonoporation has shown application potential in vivo due to its specificity, deep penetration and low cost [START_REF] Tachibana | Induction of cell-membrane porosity by ultrasound[END_REF]. Sonoporation was successfully used to transfect insulin-producing cells in a diabetic model in vivo (X. F. [START_REF] Yang | Direct reprogramming of hepatocytes into insulinproducing cells for anti-diabetic treatment by ultrasound-targeted microbubble destruction enhanced hydrodynamic gene delivery[END_REF]. Nevertheless, this technique has some limitations regarding transfection efficiency and the standardization of the sonoporation protocol as it involves many key parameters as acoustic pressure and pulse repetition frequency that vary greatly according to the acoustic equipment used [START_REF] Du | Sonoporation-Mediated Gene Transfection: A Novel Direction for Cell Reprogramming In Vivo[END_REF][START_REF] Nakagawa | Ultrasuperovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice[END_REF].

Other physical delivery strategies include the mechanical cell deformation, hydrodynamic injection and induced transduct ion by osmocytosis and propanebetaine (iTOP) (C. Liu et al., 2017).

➢ Viral delivery methods:

Over the past three decades, viral vectors have been extensively developed for delivering different nucleic acid-based therapeutics, and some of them have already been approved for clinical applications. Despite concerns regarding their safety and the potential introduction of unwanted mutations, viral delivery systems have remained the most efficient systems for delivering plasmid-based nucleic acids to mammalian cells both in

A B

vitro and in vivo. As a result, viral carriers have been extensively employed to transport plasmid-mediated CRISPR-Cas9 into cells of mammals. (C. Liu et al., 2017). Main viral delivery methods for CRISPR-Cas9 are lentivirus, adenovirus, adeno-associated virus and virus like particles.

Adeno-associated virus (AAV):

AAV is a small (20-25 nm in diameter) single-stranded DNA virus from the Dependovirus genus and Parvoviridae family that has been widely used for gene therapy. AAV is a highly effective gene therapy tool for several reasons. First and foremost, AAV has not been associated with any diseases in humans, making it a safe option for gene delivery. Moreover, there are many known serotypes of AAV, which enables the targeting of various cells with different specificities. AAV is highly efficient in delivering genes to cells, with moderate innate or adaptive immune response or associated toxicity, especially upon the first treatment with a serotype [START_REF] Daya | Gene therapy using adeno-associated virus vectors[END_REF].

Over time, the capsid of AAV could trigger immune responses, leading to potential CD-8 T-cell toxicity [START_REF] Samulski | AAV-mediated gene therapy for research and therapeutic purposes[END_REF]. However, the various AAV serotypes with broad tropism can often avoid this issue of an immune response to AAV. Another advantage of using AAV for gene therapy is that it offers a persistent source of the provided DNA, as the delivered genomic material can exist in cells indefinitely, either as exogenous DNA or by direct integration into host DNA [START_REF] Deyle | Adeno-associated virus vector integration[END_REF]. However, the advantage or disadvantage of the persistent source of DNA depends on the specific desired goals. The use of AAV particles is highly versatile, as they can be applied in in vitro, ex vivo, and in vivo experiment, making them a highly flexible delivery system.

A challenge in AAV-mediated CRISPR-Cas9 delivery is the packaging limit of AAV, which is approximately 4.5 kb, while the sequence encoding the SpCas9 protein and sgRNA is around 4.2 kb. To overcome this limitation, one solution is to use a truncated SpCas9 or SaCas9, which are smaller in size but have similar gene editing efficiency as the full-length SpCas9. However, using truncated Cas9 may lead to lower activity, around 50%, which is a drawback [START_REF] Cong | Multiplex Genome Engineering Using CRISPR/Cas Systems[END_REF]. To overcome the packaging limit of a single AAV, an alternative approach is to use dual AAVs for delivering Cas9-encoding DNA and sgRNA separately [START_REF] Swiech | In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9[END_REF].

The major limitations of AAV include the potential for the immune system to recognize and respond to the virus, which can limit the effectiveness of the therapy. Additionally, liver toxicity can occur, which can be a concern for patients with liver disease. Prolonged expression of the Cas9/gRNA complex can also be an issue, as it can lead to off-target effects and unintended mutations. Finally, integration of the viral genomic sequence into the host genome can occur, which can potentially disrupt important genes and cause adverse effects.

Adenovirus (AdV):

Adenoviruses (AdVs) in humans are characterized by a non-enveloped structure and a capsid with an icosahedral shape, measuring approximately 90-100 nm in diameter. These viruses carry a genome composed of double-stranded DNA with a size range of 26-45 kb. Adenovirus vectors have become a highly promising tool for gene delivery development due to several reasons: (1) their ability to efficiently transduce a wide variety of tissues, (2) the ease with which they can be produced in high titers, (3) their low pathogenicity in immunocompetent individuals, (4) their large cargo capacity of up to 38 kb, and (5) their ability to remain as episomal DNA without integrating into the host genome [START_REF] Crystal | Adenovirus: The first effective in vivo gene delivery vector[END_REF].

Although AdV vectors offer remarkable features, their immunogenicity remains a significant concern. While this feature is favorable for vaccine development, it poses a potential risk, such as anaphylaxis, in the case of gene delivery [START_REF] Crystal | Adenovirus: The first effective in vivo gene delivery vector[END_REF]. The high immunogenicity of AdV can result in attenuated delivery efficiency from the second injection onward, making repeated administrations ineffective (C. S. [START_REF] Lee | Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine[END_REF]. Furthermore, nearly 90% of the human population has pre-existing antibodies against AdV, which is why the AstraZeneca COVID-19 vaccine is based on chimpanzee AdV. To date, in spite of the wide use of adenoviral gene therapy in clinical trials, there is no report of the AdV vector being used for genome editing in a clinical trial.

Lentivirus (LV):

Lentiviruses (LVs) are viruses with an enveloped structure and a diameter of 80-120 nm, containing two copies of a single-stranded RNA genome of 9 kb. After reverse transcription, the RNA is converted into double-stranded DNA, which integrates into the host chromosome. LV vectors have been mostly used for ex vivo gene therapy purposes, such as stable integration of CAR (chimeric antigen receptor) cDNA into T cells. LVs have various features, including a large genetic capacity of up to 8 kb, broad tropism due to VSV-G pseudotyping, and the ability to infect both dividing and non-dividing cells [START_REF] Shuji Kubo | A New Hybrid System Capable of Efficient Lentiviral Vector Production and Stable Gene Transfer Mediated by a Single Helper-Dependent Adenoviral Vector[END_REF]. Moreover, LVs have lower immunogenicity than AAV or AdV, as they are covered by a cellular lipid-bilayer instead of a viral capsid.

Despite these advantageous features, LVs are not ideal for delivering CRISPR-Cas9 due to their integrating and long-lasting nature of transgene expression. The random integration of viral DNA may activate oncogenes or suppress tumor suppressor genes, leading to oncogenesis. Additionally, stable expression of Cas9 and gRNA may increase the risk of off-target mutagenesis. Another concern associated with LVs and integrasedefective LV (IDLVs) is the potential for replication-competent lentiviruses (RCLs) to form through recombination with wild-type HIV in HIV-infected individuals. To reduce the likelihood of RCL generation, a minimal number of LV vector-producing genes are separately coded onto multiple plasmids with minimal homology sequences [START_REF] Segall | Characterization and detection of artificial replication-competent lentivirus of altered host range[END_REF].

Virus-like particles (VLP):

Researchers have developed a virus-like particle (VLP) system based on lentiviral vectors (LV) to overcome the limitations associated with LV vectors [START_REF] Nooraei | Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers[END_REF]. VLPs are formed through the self-assembly of viral structural proteins, giving them a similar appearance to native viruses. However, VLPs lack a viral genome and are entirely replication-incompetent [START_REF] Chung | Viral nanoparticles for drug delivery, imaging, immunotherapy, and theranostic applications[END_REF]. Although VLPs have a short lifetime and are thus not suitable for long-term gene therapy, they have potential for various applications.

Recently, there has been a growing interest in VLP due to its potential for vaccine development [START_REF] Donaldson | Virus-like particle vaccines: immunology and formulation for clinical translation[END_REF] and transient delivery of the CRISPR-Cas9 system for genome editing therapy. However, caution must be exercised while using VLPs, as they have been shown to exhibit high immunogenicity and antigenicity, potentially eliciting significant cellular and humoral immunity [START_REF] Mohsen | Major findings and recent advances in virus-like particle (VLP)-based vaccines[END_REF].

Several VLP systems have been developed, including Cas9P LV, which pre-packages Cas9 protein in lentiviral particles [START_REF] Choi | Lentivirus pre-packed with Cas9 protein for safer gene editing[END_REF], VEsiCas, which passively incorporates SpCas9 [START_REF] Montagna | VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9[END_REF], NanoBlades, which fuse SpCas9 with retroviral Gag (Mangeot et al., 2019), and Gesicle system, which uses dimerization-based incorporation of SpCas9 [START_REF] Campbell | Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus[END_REF].

Virus-like particle-based platforms pose additional challenges that need to be carefully addressed. Due to the absence of genetic material, VLPs are prone to instability, and they are more complex and costly to produce [START_REF] Mohsen | Interaction of viral capsid-derived virus-like particles (VLPs) with the innate immune system[END_REF]. Impurities, such as host cell debris, proteins, and DNA, are a significant concern during downstream processing, as they can cause unintended side effects if not removed correctly [START_REF] Van Oers | Opportunities and challenges for the baculovirus expression system[END_REF].

Additionally, the assembly process can be difficult, as genetic fusion of epitope sequences into VLPs may lead to particle misfolding or loss of self-assembly property [START_REF] Guo | Immunogenicity of a viruslike-particle vaccine containing multiple antigenic epitopes of toxoplasma gondii against acute and chronic toxoplasmosis in mice[END_REF].

➢ Non-Viral delivery methods:

In recent times, nanomaterials have increasingly demonstrated distinctive benefits in gene delivery compared to viral vectors. Various nano-delivery systems have been created for CRISPR-Cas9, including cationic liposomes, lipid nanoparticles (LNPs), cationic polymers, vesicles, and gold nanoparticles. The forthcoming section will develop deeper into these non-viral delivery methods, providing more comprehensive details.

Exploring nanoparticles as CRISPR-Cas9 delivery systems

The European Commission defines nanoparticles as materials comprising particles, either naturally occurring, incidental, or manufactured, that exist as unbound entities, aggregates, or agglomerates. These nanoparticles have at least one external dimension ranging from 1 to 100 nanometers in more than 50% of the particle population [START_REF] Potočnik | Recommendation of 18 October 2011 on the definition of nanomaterial[END_REF]. Nanoparticles can be classified into any of various types, according to their size, shape, and material properties. Some classifications distinguish between organic and inorganic nanoparticles; the first group includes dendrimers, liposomes, and polymeric nanoparticles, while the latter includes fullerenes, quantum dots, and gold nanoparticles (Figure 17).

Figure 17. Nanoscale integration of biomolecules and main nanoparticles types [START_REF] Saallah | Nanoparticles carrying biological molecules: Recent advances and applications[END_REF] Lipid NPs Gold NPs Nanoclews Polymeric NPs Peptide NPs

Critical challenges for NP mediated CRISPR-Cas9 delivery

The unique properties of nanoparticles and their behavior in biological environment enable exciting and integrative approaches to study fundamental biological questions. They appear as interesting carriers for CRISPR-Cas9 system. It has to be noticed that CRISPR-Cas9 delivery by nanoparticles encounters various obstacles that must be overcome to achieve successful delivery to the intended tissues (Figure 18). NPs must protect their cargo from destabilizing serum components, enzyme degradation, and clearance by immune cells, as well as liver and kidneys clearance. Indeed, after reaching the target cells, nanoparticles need to traverse the cell membrane to gain entry. If they are internalized by the cell through endocytosis, NPs face the challenge of escaping from degradative endolysosomal compartments. In the case of delivering mRNA, the cargo must be released in the cytosol, allowing ribosomes to carry out translation. In DNA delivery, the plasmid DNA must be transported to the nucleus where it is transcribed into mRNA. Subsequently, the mRNA transcript should enter the cytosol and undergo translation into protein. Whether protein complexes are synthesized internally by the cell itself or directly delivered through the nanocarrier, they must ultimately reach the nucleus in order to enable gene editing. Finally, the CRISPR-Cas9 system needs to overcome the nuclear barrier in order to exhibit its gene-editing activity. 2) escape from degradative endolysosomal compartments. In the case of mRNA delivery, cargo release must occur in the cytosol (4a) to enable translation by ribosomes (5a). In DNA delivery, plasmid DNA needs to be transported to the nucleus (4b) for transcription into mRNA (5b), which must then enter the cytosol to undergo translation into protein (6b). Protein complexes, whether synthesized within the cell (6a, 7b) or delivered directly by the nanocarrier (4c), must localize to the nucleus for gene editing to take place. The common intracellular delivery steps applicable to all cargo formats are represented by black arrows, while red, green, and blue arrows indicate steps specific to mRNA, plasmid DNA, and protein complexes, respectively [START_REF] Rui | Non-Viral Delivery To Enable Genome Editing[END_REF].

Important characteristics of nanoparticle-based delivery systems

Several key characteristics of nanoparticles influence their performance as drug carriers, including particle size, surface properties, drug loading, drug release, and targeted drug release, morphology and purity (Figure 19). These characteristics play a crucial role in determining the efficacy, safety, and selectivity of nanoparticle-based delivery systems. Understanding and optimizing these properties are essential for the successful design and implementation of nanoparticle-based delivery strategies.

Figure 19. Important characteristics of nanoparticles as delivery systems. Adapted from [START_REF] Todaro | Characterization and Functionalization Approaches for the Study of Polymeric Nanoparticles: The State of the Art in Italian Research[END_REF].

Particle Size: The size distribution of nanoparticles is a critical characteristic for drug delivery. It plays a crucial role in determining their in vivo distribution, biological fate, toxicity, and targeting ability. Furthermore, it can impact important aspects such as drug loading, drug release, and nanoparticle stability (He et al., 2010). By carefully controlling the particle size, researchers can optimize the performance and effectiveness of these drug delivery systems, ensuring efficient delivery of therapeutic agents to the intended sites and minimizing any potential adverse effects. Their small size enables them to penetrate biological barriers, such as cell membranes and tissues, facilitating efficient delivery to target sites. Additionally, nanoparticles with appropriate sizes can exploit the

Important features of Nanoparticles

enhanced permeability and retention (EPR) effect, selectively accumulating in tumors or inflamed tissues.

Surface Properties:

The surface properties of nanoparticles play a vital role in their interaction with biological systems. By modifying the surface of nanoparticles, their stability, biocompatibility, and cellular uptake can be optimized. In addition to nanoparticle size, the hydrophobicity of nanoparticles plays a crucial role in their interaction with blood components, such as opsonins, which bind to the nanoparticle surface. Opsonization is an immune process which uses opsonins to tag foreign pathogens for elimination by phagocytes. It is crucial to minimize opsonization and extend the circulation time of nanoparticles. This can be accomplished by coating nanoparticles with hydrophilic polymers or surfactants, or by formulating nanoparticles using biodegradable copolymers that possess hydrophilic properties, such as polyethylene glycol (PEG) [START_REF] Singh | Sotorasib for Lung Cancers with KRAS p.G12C Mutation[END_REF]. By employing these strategies, nanoparticles can evade the immune system and remain in circulation for longer periods, thereby increasing the chances of successful delivery.

The surface charge: It plays also a crucial role in NPs efficacy. Positively-charged NPs, owing to their interaction with negatively charged cell membranes, exhibit enhanced cellular binding and uptake in a non-specific manner (F. [START_REF] Zhao | Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials[END_REF]. While a positively charged drug delivery system (DDS) may enhance therapeutic efficacy in vitro, it can lead to rapid binding to vascular endothelial cells, resulting in reduced accumulation of the drug in tumors in vivo. Similarly, highly negatively charged nanoparticles (NPs) are quickly taken up by the reticuloendothelial system (RES) in the liver and spleen. Therefore, it is advisable to develop neutral or slightly negatively charged NPs.

Drug Loading: An effective nanodelivery system should possess a significant loading capacity for maximizing therapeutic efficacy. Nanoparticles can encapsulate drugs within their core, adsorb them onto their surface, or chemically conjugate them to the nanoparticle matrix. The high surface area-to-volume ratio of nanoparticles allows for a large drug payload, enhancing the drug loading capacity. Effective drug loading ensures the stability of the encapsulated drugs, protects them from degradation, improve its bioavailability and facilitates their controlled release at the target site.

Drug Release: Controlled drug release is a key feature of nanoparticles for drug delivery.

Nanoparticles can be engineered to release drugs in a controlled manner, allowing for sustained and controlled drug release over a desired period. This enables optimal drug concentrations at the target site, reducing the need for frequent dosing and minimizing potential side effects. This is particularly beneficial for drugs with narrow therapeutic indices or high toxicity profiles. Controlled drug release can be achieved by tailoring the composition, structure, and surface properties of nanoparticles, as well as through external stimuli, such as pH, temperature, or light (C. [START_REF] Li | Functional Nanoparticles for Enhanced Cancer Therapy[END_REF].

Targeted Drug Delivery: Targeted drug delivery is a significant advantage of nanoparticles. By functionalizing nanoparticles with targeting ligands, they can selectively accumulate at specific target sites, such as tumors or inflamed tissues. Targeted delivery can be accomplished through active or passive means. Active targeting involves the conjugation of the therapeutic agent or carrier system with a tissue or cellspecific ligand to facilitate specific binding [START_REF] Bertrand | Cancer Nanotechnology: The impact of passive and active targeting in the era of modern cancer biology[END_REF][START_REF] Morales-Cruz | Smart targeting to improve cancer therapeutics[END_REF]. On the other hand, passive targeting involves the incorporation of the therapeutic agent into a macromolecule or nanoparticle that naturally reaches the target organ without specific targeting mechanisms. This targeted approach improves drug efficacy by concentrating the therapeutic agent at the desired location while minimizing exposure to healthy tissues. Targeted drug delivery also allows for reduced drug dosage, limiting systemic toxicity and enhancing the therapeutic index. It enables precision medicine and personalized treatment approaches, maximizing therapeutic outcomes.

Nanocarriers as optimal platforms for precise CRISPR-Cas9 delivery

Nanoparticles have emerged as promising carriers for CRISPR-Cas9 delivery due to their unique properties mentioned before, such as high stability, tunable surface characteristics, and ability to protect and efficiently transport RNA molecules to target cells. Among the various types of nanoparticles used for CRISPR-Cas9 delivery, lipidbased delivery systems, polymer-based delivery systems, gold nanoparticles and exosomes are the most currently used, as listed in the table below (Table 3).

Table 3. Overview of nanoparticles used for CRISPR-Cas9 delivery in the literature and their characteristics [START_REF] Duan | Nanoparticle Delivery of CRISPR/Cas9 for Genome Editing[END_REF].

Lipid nanoparticles (LNP)

Lipid-based nanoparticles are widely employed as the primary delivery system for CRISPR-Cas9. In these lipid-based systems, cationic lipids are typically utilized to encapsulate anionic Cas9 DNA, mRNA, or protein through electrostatic interactions. This encapsulation ensures the protection of the CRISPR/Cas9 system from degradation and minimizes the organism's immunological response. LNP also facilitates CRISPR-Cas9 uptake by endocytosis.

A range of cationic lipid-assisted nanoparticles (CLANs) based on PEG-block-poly(D,Llactic-co-glycolic acid) (PEG-b-PLGA) have been developed (Figure 20). These CLANs carry a macrophage-specific promoter (CD68) that drives the expression of Cas9 plasmid. By utilizing sgRNA targeting the Ntn1 gene, the CLANs specifically edit the Ntn1 gene in macrophages, resulting in reduced netrin1 expression and improved symptoms of type II diabetes in vivo [START_REF] Luo | Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles[END_REF]. However, the editing efficiency of using LNPs to package CRISPR-Cas9 plasmids has not met the clinical requirements because their delivery efficiency to primary cells or in vivo animal experiments is low. promoter, thus is specifically expressed in macrophages. After intravenous injection in mouse, nanoparticles could be endocytosed by diverse cells, including tissue cells, T cells, B cells, neutrophils, monocytes, and macrophages. However, the CD68 promoter ensured the Cas9 expression occurred only in monocytes and macrophages and led to gene knockdown [START_REF] Luo | Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles[END_REF].

Polymeric NPs

Compared with cationic lipid carriers, cationic polymer carriers show chemical diversity and functional potential, providing more choices for flexible structural designs. Cationic polymer nanoparticles have been widely used to deliver different nucleic acid types, including plasmid DNA and mRNA. Cationic polymers, such as polyethyleneimine and chitosan, were the most frequently used carriers for CRISPR/Cas9 delivery. Similar to lipid carriers, polymer nanoparticle carriers can traverse the membrane through endocytosis and protect the loaded cargoes from the immune response and nuclease degradation. The polymers provide protection to the RNA cargo and can be engineered to optimize the release of RNA within target cells. Additionally, the surface of polymerbased nanoparticles can be modified to improve their stability, cellular uptake, and targeting capabilities.

For instance, a remotely guided near-infrared imaging-controlled CRISPR/Cas9 genome editing strategy triggered by near-infrared (NIR-II) light is reported, based on a rationally designed semiconducting polymer brush (SPPF) (Figure 21). The SPPF not only serves as a vector for delivering CRISPR/Cas9 cassettes but also controls endolysosomal escape and payload release through photothermal conversion under laser irradiation. When exposed to the laser, the SPPF and CRISPR/Cas9 cassette nanocomplex induces precise, efficient, and site-specific genome editing, both in vitro and in vivo, with minimal toxicity (L. [START_REF] Li | A Rationally Designed Semiconducting Polymer Brush for NIR-II Imaging-Guided Light-Triggered Remote Control of CRISPR/Cas9 Genome Editing[END_REF].

Gold NPs

A novel nanocarrier called CRISPR-Gold has been developed by Lee et al, and consists of gold nanoparticles, Cas9 RNP, donor DNA, and an endosomal disruptive polymer known as poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (K. Lee et al., 2018). This nanocarrier employs endocytosis to enter cells and then disrupts the endosomal membrane, releasing its cargo into the cytoplasm. Within the cytoplasm, DNA is liberated from the gold core of CRISPR-Gold by the action of cytosolic glutathione, enabling effective editing of the target gene. In vivo experiments have demonstrated the capabilities of CRISPR-Gold, revealing its ability to correct dystrophin mutations in mdx mice with minimal off-target damage (Figure 22).

Exosomes

In addition to other delivery methods, exosomes have the ability to transport gRNA and Cas9 proteins to target cells, effectively enabling the functional delivery of the CRISPR-Cas9 system (R. [START_REF] Chen | Friend or Foe? Evidence Indicates Endogenous Exosomes Can Deliver Functional gRNA and Cas9 Protein[END_REF]. These exosomes are natural vesicles surrounded by a membrane and possess notable advantages such as high biocompatibility and minimal immunogenicity. By taking advantage of their exceptional biocompatibility and the prompt degradation of gRNA and Cas9 proteins within cells, the delivery of gRNA and Cas9 helps mitigate the risks associated with off-target effects and integration into the genome.

Peptide based nanoparticles (PBN)

Peptide-based delivery systems have displayed immense promise in delivering nucleic acids and particularly CRISPR-Cas9 components. Cationic peptides offer the potential to form complexes with a larger cargo capacity compared to viral systems. They also enable intracellular delivery while minimizing immune or cytotoxic responses. One notable type of peptide is the cell-penetrating peptide (CPP), which consists of short peptides capable of directly interacting with cell membranes. This interaction allows for the successful intracellular delivery of therapeutic cargos by traversing cell membranes. The utilization of CPPs in this manner opens up the possibility of conducting multiple precise genome engineering rounds using the CRISPR-Cas9 system. This approach has the potential to advance the development of next-generation cell therapies, incorporating precise knockin and knockout edits. The subsequent section will develop in further details this approach.

Unraveling the remarkable potential of peptide-based nanoparticles (PBN) as delivery system

Cell penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), are short peptides consisting of 6 to 30 amino acids. They can either be synthetic or naturally occurring and possess the remarkable ability to transport a wide range of cargo across cellular membranes while maintaining their functionality. Since their initial discovery and characterization, the field of CPPs as delivery vectors has experienced significant growth.

CPPs have demonstrated their versatility in delivering various types of cargo. These cargoes include small peptides, complete proteins, nucleic acids such as RNA and DNA, as well as imaging agents like radioisotopes and fluorescent probes. The potential applications of CPPs in diverse fields such as drug delivery, gene therapy, and imaging have prompted extensive research and development efforts.

At first, cell penetrating peptides were primarily utilized by directly linking them to nucleic acids (NAs) through covalent conjugation. However, there has been a growing trend towards non-covalent approaches that leverage the attractive forces of electrostatic and hydrophobic interactions between CPPs and nucleic acids (Figure 23). As a result of these interactions, peptides and nucleic acids were able to spontaneously assemble when incubated together, leading to the formation of peptide-based nanoparticles (PBNs). Depending on incubation conditions and nature of the peptides, they can form naked PBNs, multi-grafted PBNs, or prospective micelle-like PBNs. These nanocarriers are able to penetrate the cell membrane either by direct translocation or by endocytosis and then release the cargo into the targeted cell.

This breakthrough allowed peptides to enter the realm of nanomedicine, offering promising applications [START_REF] Boisguérin | Delivery of therapeutic oligonucleotides with cell penetrating peptides[END_REF]. Furthermore, peptide-based vectors have gained recognition as viable options for delivering therapeutic nucleic acids. Their advantages include straightforward automated synthesis, simple formulation processes, and biocompatible characteristics, making them highly suitable for this purpose.

Within the wide range of CPP, some amphipathic peptides have been specifically designed to possess both hydrophilic and hydrophobic regions. This enables them to effectively interact with NAs and cellular membranes. Primary amphipathic CPPs have hydrophilic and hydrophobic domains distributed along the peptide chain, while secondary amphipathic peptides form these domains through the folding of their secondary structure [START_REF] Koltun | Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery[END_REF][START_REF] Sharma | Insight into the role of physicochemical parameters in a novel series of amphipathic peptides for efficient DNA delivery[END_REF]. Secondary amphipathic peptides have been utilized to create PBNs. The ability of CPPs to form PBNs is associated with various structural properties. The conformational state of non-covalent CPPs, along with physicochemical parameters such as amphipathicity, charges, and specific residues, directly influences the efficiency of PBNs in NA delivery [START_REF] Konate | Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide[END_REF][START_REF] Morris | A new peptide vector for efficient delivery of oligonucleotides into mammalian cells[END_REF].

Building upon the chimerical peptide carrier PPTG1, known for its nucleic acid interaction but lacking affinity for short oligonucleotides, researchers engineered the CADY peptide (Ac-GLWRALWRLLRSLWRLLWRA-Cya) [START_REF] Multiplex Crombez | A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells[END_REF]. CADY, a secondary amphipathic peptide comprising 20 residues, underwent strategic modifications to improve its stability and boost cellular uptake. Characterized by a high positive charge and moderate hydrophobicity, CADY features four Trp groups and five Arg residues. It interacts strongly with membrane lipids, adopting a helical conformation that aids in cellular uptake. CADY forms stable complexes with small oligonucleotides and siRNA, facilitating their efficient uptake by various cell lines. The peptide's interaction with siRNA is driven by electrostatic and specific amino acid contacts and Trp residues have a crucial function in enhancing the stability of the complexes and aiding in their integration into the membrane. CADY-mediated delivery of siRNA leads to significant knockdown of the target gene without toxicity [START_REF] Multiplex Crombez | A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells[END_REF]. It enters cells independently of the major endosomal pathway. The unique properties of CADY suggest its potential for impactful siRNA-based applications.

PBN are novel vectors for CRISPR-Cas9 delivery

Possibilities of CPP-mediated delivery of Cas9 RNPs have been explored in several studies where covalent approaches were used to conjugate the Cas9 endonuclease and CPP. In 2014, Ramakrishna et al. introduced PBNs as a delivery system for CRISPR-Cas9. Their study demonstrated that treating human cell lines with cell-penetrating peptide (CPP)conjugated recombinant Cas9 protein and CPP-complexed guide RNAs resulted in endogenous gene disruptions, specifically targeting the CCR5 gene. T7E1 assay showed gene editing efficiencies between 3% and 16% [START_REF] Ramakrishna | Gene disruption by cellpenetrating peptide-mediated delivery of Cas9 protein and guide RNA[END_REF].

Lostale-Seijo et al. introduced in 2017 a novel supramolecular approach for delivering Cas9 using an amphiphilic penetrating peptide. This strategy involved the formation of a hydrazone bond between a cationic peptide scaffold and a hydrophobic aldehyde tail. The findings from their research demonstrated that peptide amphiphilic vectors could effectively deliver Cas9 in a single incubation step, exhibiting gene editing efficiency between 20 and 40% and while inducing minimal toxicity [START_REF] Liu | Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition[END_REF].

One year later, Wang et al. presented their work on PEGylated peptide nanoparticles (referred to as P-HNPs) (Figure 24). These nanoparticles were based on a cationic αhelical polypeptide, poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-Lglutamate), and were designed for delivering Cas9 expression plasmid and sgRNA to different cell types and gene-editing scenarios. The incorporation of the cell-penetrating α-helical polypeptide facilitated cellular uptake and facilitated the release of pCas9 and/or sgRNA from the endosome, enabling their transport into the nucleus. The P-HNPs displayed excellent colloidal stability and achieved transfection efficiency of up to 60% for Cas9 and uptake efficiency of 67.4% for sgRNA. These results represented an improvement compared to existing polycation-based gene delivery systems. In vitro experiments demonstrated that the P-HNPs enabled single or multiplex gene editing with an efficiency of up to 47.3%. Notably, when delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene, the P-HNPs achieved a remarkable 35% gene deletion in HeLa tumor tissue, resulting in a significant reduction of Plk1 protein levels by 66.7%. This led to a substantial suppression of tumor growth by more than 71% and increased the animal survival rate to 60% within a 60-day timeframe (H. X. [START_REF] Wang | Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide[END_REF]. In 2019, Krishnamurthy demonstrated successful delivery of protein and SpCas9 or AsCas12a RNP to cultured human airway epithelial cells and mouse lungs using specially designed amphiphilic peptides. These peptides, in combination with GFP protein or CRISPR RNP, effectively entered both types of human epithelial cells in culture and mouse airway epithelia. By administering the shuttle peptides with SpCas9 or AsCas12a RNP, they achieved targeted editing of specific sites in the airway epithelia of mice. Importantly, no signs of short-term toxicity were observed, and the delivered components were primarily localized in the respiratory tract [START_REF] Krishnamurthy | Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia[END_REF].

In a recent study conducted in 2021, Gustafsson et al. repurposed the cell-penetrating peptide PepFect14 (PF14) to deliver Cas9 RNP. The RNP-CPP complex exhibited high editing rates of up to 80% in HEK293T cells, showing activity at low nanomolar concentrations with no detectable toxicity. The editing efficiency of the complex was comparable to or better than that of commercially available reagents like lipofectamine. Furthermore, the CPP-Cas9-RNP complexes demonstrated excellent stability, remaining effective even after storage under various conditions, including freeze-thaw cycles and freeze-drying. This was followed by two studies in 2023. The first one introduced an approach they called Peptide-Assisted Genome Editing (PAGE) using the CRISPR-Cas system. This technique enabled rapid and reliable editing of primary cells, such as human T cells and human hematopoietic progenitor cells, while minimizing potential toxicity. The editing efficiencies achieved in these primary cells were notably high, exceeding 98% [START_REF] Zhang | Efficient engineering of human and mouse primary cells using peptide-assisted genome editing[END_REF]. In a second study this year, Foss et al. demonstrated a significant increase in the yields of edited primary human lymphocytes by delivering a CRISPR RNP mixed with an amphiphilic peptide identified through screening. They assessed the efficacy of this straightforward delivery method by targeting gene knockouts in various immune cell types, including T cells, B cells, and natural killer cells. The delivery of Cas9 or Cas12a RNPs, as well as an adenine base editor, enabled efficient gene editing in these cells. This study highlighted the potential of utilizing amphiphilic peptides for enhancing gene editing outcomes in primary human lymphocytes [START_REF] Foss | Peptidemediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes[END_REF].

Peptide-based nanoparticles have emerged as promising delivery systems for CRISPR-Cas9 due to their unique properties and demonstrated effectiveness. The use of PBN allows for the efficient delivery of CRISPR-Cas9 components into target cells, leading to precise gene editing. However, it is worth noting that these nanocarriers have been predominantly employed for the delivery of CRISPR-Cas9 in DNA or RNP forms, rather than mRNA form. This may arise from the challenges associated with the larger size and stability of mRNA molecules. Nonetheless, the versatile and customizable nature of peptide-based nanoparticles, along with their ability to enhance cellular uptake and protect the cargo, makes them a promising avenue for advancing the delivery of CRISPR-Cas9 under mRNA form and expanding its applications in gene therapy and biomedical research. However, most therapeutic strategies are still based on DNA or RNP modes, with mainly ex vivo approaches. Therefore, further research and development efforts are needed to optimize the delivery of mRNA-based CRISPR-Cas9 delivery and in vivo applications still require optimization.

CRISPR/Cas systems have recently gained widespread usage in research to develop potential therapeutic tools for various human diseases. These include genetic disorders like cystic fibrosis and muscular dystrophy, infectious diseases such as HIV and malaria, and complex ailments like cancer. In particular, numerous studies in the field of cancer research have employed CRISPR/Cas systems with the objective of targeting inactivated tumor suppressor genes or hyperactive oncogenes, which are critical events in tumor progression. In the subsequent section, I will concentrate specifically on the Kirsten rat sarcoma virus oncogene (KRAS) that is one of the main tumoral target selected by the company Divincell with which I did my thesis. It is worth noting that despite extensive efforts thus far, KRAS has proven challenging to target using conventional molecular therapy approaches. However, the CRISPR/Cas9 system presents an exciting new avenue for editing KRAS mutations in cancer cells, offering a novel molecular opportunity for intervention.

Decoding the KRAS oncogene

KRAS is the most commonly mutated oncogenic protein in solid tumors. It is one of the main actors in the regulation of cell-cycle pathways. However, due to the absence of pharmacologically targetable pockets within the mutant isoforms, it has historically been considered as "undruggable." Nevertheless, advances in drug design have resulted in the development of inhibitors that are selective for mutant KRAS in either its active or inactive state. These inhibitors have demonstrated efficacy in patients with specific KRAS mutations linked to cancer and have led to significant changes in medical practice. Despite these breakthroughs, drug resistance remains a challenge, which restricts the depth and/or duration of responses to these agents.

Discovery of KRAS

In 1964, Dr. Jennifer Harvey observed the rapid onset of sarcoma in rats infected with Moloney's leukemogenic virus (MLV). This was followed by Werner Kirsten's discovery of similar transformation in 1967 upon inoculation of a murine erythroblastosis virus (Figure 25) [START_REF] Kirsten | Morphologic responses to a murine erythroblastosis virus[END_REF]. These retroviral genes responsible for transformation were named Ha-ras and Ki-ras, respectively, with the term 'ras' being an acronym for "rat sarcoma". The protein product of ras, a 21 kDa polypeptide called p21, was identified in 1979 [START_REF] Shih | Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus[END_REF], which facilitated understanding of its function, particularly its high affinity to bind guanine-containing nucleotide. The term 'oncogene,' though appeared as early as 1969 [START_REF] Huebener | ONCOGENES OF RNA TUMOR VIRUSES AS DETERMINANTS OF CANCER[END_REF], gained traction when these transforming ras genes were identified in human cancers. In 1982, HRAS and KRAS, the human counterparts of Ha-ras and Ki-ras, were discovered in human bladder and lung carcinoma cell lines [START_REF] Der | Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kristen sarcoma viruses[END_REF]. The third member of the ras family, NRAS, was identified the following year in a neuroblastoma cell line [START_REF] Hall | Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1[END_REF]. In 1984, a KRAS G12R point mutation was first identified in the tumor tissue of a 66-year-old man with squamous cell lung carcinoma, which was not present in normal parenchymal or lymphocytes [START_REF] Santos | Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient[END_REF]. The KRAS G12C mutation was identified in the same year in a human lung tumor (PR371) that was propagated in nude mice [START_REF] Nakano | Isolation of transforming sequences of two human lung carcinomas: Structural and functional analysis of the activated c-K-ras oncogenes[END_REF].

These pioneering studies highlighted the pivotal role of KRAS oncogene in driving human carcinogenesis, sparking intense research efforts towards developing effective therapeutic strategies and RAS inhibitors. Despite decades of investigation, progress in this field has been hindered by the perceived "undruggable" nature of KRAS. However, in 2021, the first KRAS inhibitor, targeting a specific KRAS mutation (G12C) was FDAapproved, marking a significant milestone in the development of targeted therapy for KRAS-driven cancers and heralding a new era in cancer treatment. [START_REF] Nakajima | FDA Approval Summary: Sotorasib for KRAS G12C-Mutated Metastatic NSCLC[END_REF]. This was rapidly followed by the approval of adagrasib, another G12C inhibitor, in December 2022 [START_REF] Dhillon | Adagrasib: First Approval[END_REF].

Navigating the biology of KRAS

The family of Rat sarcoma (RAS) oncogenes consists of three members, namely KRAS, HRAS, and NRAS, which have a significant role in human cancer. These genes encode 21 kDa monomeric GTPases that play a crucial role in transmitting extracellular signals to intracellular signal transduction cascades. As part of the small GTPase superfamily, KRAS proteins exhibit exclusive binding to GTP (G proteins).

Few forms of KRAS exist. KRAS-1 is a pseudogene, meaning that it is a non-functional copy of the KRAS gene that has accumulated mutations over time and is unlikely to produce a functional protein. KRAS-1 is situated on the short arm of chromosome 6. On the other hand, KRAS-2 is the primary KRAS gene. It is located on the short arm of chromosome 12 and produces a functional KRAS protein [START_REF] Popescu | Chromosomal localization of three human ras genes by in situ molecular hybridization[END_REF]. The KRAS-2 coding region extends across six exons and measures over 45 kB (Figure 26). Alternative splicing on its fourth exon leads to the production of two protein isoforms, namely KRAS-4A and KRAS-4B, with 188 and 189 amino acid sequences, respectively [START_REF] Capon | Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations[END_REF]. In both clinical and research settings, KRAS typically refers to KRAS-4B, which is the primary transcriptomic product found in human cells.

The KRAS protein is composed of two domains: the N-terminal catalytic domain (Gdomain), responsible for guanine binding, and the hypervariable region (HVR), located at the C-terminal (Figure 26). The catalytic domain is a region that is highly conserved and exhibits a high degree of homology. It comprises the P-loop, switch I, and switch II regions. The G-domain acts as a GTP-GDP switch, facilitating GTP-GDP exchange [START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]. The P-loop serves as the region for phosphate binding and stabilizes the nucleotide phosphates, whereas the switch regions form binding surfaces for effector proteins. The switch in the G-domain is regulated mainly by guanine exchange factors (GEFs) that promote GDP to GTP exchange and activation, and by deactivating of factors such as GTPase activating proteins (GAPs).

The C-domain, responsible for anchoring RAS to the inner surface of the plasma membrane, exhibits a high degree of variability. It contains the CAAX box, consisting of cysteine, two aliphatic amino acids, and another residue, which undergoes posttranslational modifications such as prenylation [START_REF] Kato | Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity[END_REF].

The most common activating mutations of KRAS (codons 12, 13, 61, 117, and 146) are clustered around the nucleotide binding pocket. G12 and G13 are positioned on the Ploop, which is essential to stabilize the nucleotide in the active site, but they have distinct effects on KRAS biochemistry. Codon 12 mutants generally affect both intrinsic and GAPinduced hydrolysis, without altering the rate of nucleotide exchange, while codon 13 mutants affect hydrolysis and increase intrinsic exchange by approximately 10-fold [START_REF] Hunter | Biochemical and structural analysis of common cancer-associated KRAS mutations[END_REF][START_REF] Smith | NMR-based functional profiling of RASopathies and oncogenic RAS mutations[END_REF].

The potential mutation site Q61 is located at the N-terminus of switch II, where it participates in the conformational changes associated with this region during the interconversion between structural states. The hydrocarbon portion of the K117 side chain packs against the guanine ring of the nucleotide, and the charged amine group of K117 forms a salt-bridge with the backbone carbonyl of G13 in the P-loop, playing a dual role in stabilizing the nucleotide and promoting hydrolysis. A146 likely contributes to nucleotide specificity, as it is located in a tightly packed space near the guanine base of the nucleotide. Mutations at A146 promote nucleotide exchange (1,000-fold increase over WT) in the absence of guanine nucleotide exchange factor but do not affect intrinsic GTPase activity [START_REF] Feig | Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins[END_REF].

Figure 26. KRAS gene and mutational hotspot [START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

KRAS mediated signaling pathway

The activation of KRAS triggers downstream signaling through three primary pathways: the MAP kinase pathway, the PI3K-AKT-mTOR pathway, and the pathways involving the tumor invasion and metastasis-inducing protein 1 (TIAM1-RAC) and RAS-related protein (RAL) (Figure 27). Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. It involves the phosphorylation of RAS, RAF (Rapidly Accelerated Fibrosarcoma), MEK (MAPK/ERK kinase), and ERK (extracellular signal-regulated kinase). The binding and phosphorylation of RAF molecules are facilitated by conformational changes brought about by RAS activation and dimerization. In the case of mutant RAS, its dimers lead to enhanced RAF binding and activation [START_REF] Takács | The effects of mutant Ras proteins on the cell signalome[END_REF], making this the major downstream signaling pathway of mutant RAS. The final enzyme in the MAPK pathway, called ERK, translocates to the nucleus, where it activates various transcription factors, promoting cellular differentiation and proliferation [START_REF] Eblen | Extracellular-Regulated Kinases: Signaling From Ras to ERK Substrates to Control Biological Outcomes[END_REF]. The activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by the GTP-RAS complex leads to the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3, in turn, phosphorylates AKT, which then triggers downstream mTOR, FOXO, and NF-kB phosphorylation, thereby promoting cell survival and resistance to apoptosis [START_REF] Vara | P13K/Akt signalling pathway and cancer[END_REF]. The PI3K pathway is frequently upregulated in RAS mutations, but the role of RAS in PI3K activation in normal cells is less clear. RAL guanine nucleotide stimulator (RALGDS) is a downstream protein that is also activated by RAS, and it promotes cell migration and RAS-dependent tumor growth. In addition, RALGDS stimulates the Jun N-terminal kinase (JNK) pathway, which leads to transcription and cell-cycle progression. TIAM1-RAC are guanine nucleotide exchange factors (GEFs) that lead to phosphorylation of PAK serine/threonine kinases, which are involved in cytoskeleton rearrangement and cell migration [START_REF] Lambert | Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism[END_REF]. Therefore, in its normal state, KRAS serves as a key link between several cell-cycle pathways, and an activated KRAS mutation leads to oncogenesis through multiple downstream activation pathways. Guanine Nucleotide Exchange Factor (GEF) proteins, which initiate the exchange of GDP for GTP. One of the GEFs, SOS1, is a RAS activator. Active, GTP-bound KRAS can trigger activation of downstream signaling molecules including RAF/MEK/ERK, PI3K/AKT/mTOR, and RAL/NF-κβ pathways, which result in the expression of genes that promote cellular survival, proliferation and differentiation. It can self-inactivate by cleaving the terminal phosphate group on the GTP, converting it to GDP. This inactivation process is accelerated by GTPase-activating (GAP) proteins. Adapted from [START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

Insights into oncogenic KRAS mutations

KRAS is the most commonly mutated gene in the RAS family and is widely recognized as a key driver of oncogenesis in human cancers, particularly in colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and non-small-cell lung cancer (NSCLC). KRAS mutations are frequently observed in pancreatic cancers (about 90%), followed by colorectal cancer (about 45%) and NSCLC (about 35%). The majority of KRAS mutations are single-base missense mutations, which are most commonly found at codons 12 (83%), 13 (14%), or 61 (2%) on exons 2 and 3. The allele distribution pattern of oncogenic KRAS mutation subtypes in various cancers reflects both environmental causes and apparent tissue selective tropism.

The two most common KRAS mutations, G12D and G12V, are observed in nearly half of all KRAS-driven cancers (Figure 28A) [START_REF] Sweeney | AACR project genie: Powering precision medicine through an international consortium[END_REF]. In the United States alone, approximately 150,000 new incidences of tumors carrying KRAS mutations are diagnosed per year, with G12D and G12V contributing to nearly 80,000 cases of PDAC, CRC, and lung adenocarcinoma (LUAD) cancer patients (Figure 28B). In PDAC, G12D and G12 are the most frequently found KRAS mutations with 37% and 28% respectively. Similarly, they are predominant in CRC with 12.5% and 8.5% frequency respectively. After a 13.6% frequency of KRAS G12C, the G12D and G12V mutations are predominant in LUAD with presence in 4.9% and 5.9% of the cases (Figure 28C).

The occurrence of KRAS mutations in human cancer, as well as evidence that activated KRAS can initiate and maintain the transformed phenotype, has led to intense efforts to discover compounds that target KRAS. Despite four decades of mostly unsuccessful research, there has been recent progress with the development of the first selective drugs against KRAS G12C, which have been quickly approved as a second-line treatment for KRAS G12C-driven non-small-cell lung cancer. However, these inhibitors will only be effective for a portion of all KRAS-mutated cancers, as the major KRAS drivers are the G12D and G12V mutations. This highlights the significant need for additional KRAS therapies in the medical field.

Main KRAS-driven cancers

Pancreatic, colorectal, and lung cancers are among the most common cancers worldwide, and while treatment options have improved, these cancers remain challenging to manage. The apparition of mutant K-ras initiates a signal transduction cascade that provides a strong progrowth signal, increases cell motility and invasion, and profoundly rearranges cell metabolism to a growth-promoting state. In pancreatic cancer, Ras mutation initiates paracrine signals that promote and maintain stromal desmoplasia, a key mediator of noncell-autonomous resistance.

Pancreatic cancer ranks as the third highest contributor to cancer-related fatalities and is characterized by a very unfavorable prognosis [START_REF] Siegel | Cancer Statistics[END_REF]. Due to the insufficiency of early detection methods and the absence of noticeable symptoms, the 5year survival rate is only 7-8% [START_REF] Ying | Genetics and biology of pancreatic ductal adenocarcinoma[END_REF]. PDAC, a highly aggressive form of cancer, is characterized by a unique fibro-inflammatory microenvironment that plays a significant role in cancer initiation and growth. The epithelial manifestation of pancreatic cancer involves the progression of hyperplastic lesions known as pancreatic intraepithelial neoplasia (PanIN) in its initial stages. Through a series of mutations, starting with KRAS, PanIN transforms into Pancreatic Ductal Adenocarcinoma (PDAC), accounting for 90% of pancreatic cancer cases, as illustrated in Figure 29. The majority of PDAC cases involve mutations in the KRAS oncogene, which not only affects the tumor itself but also influences its surrounding microenvironment. In pancreatic cancer, surgery is the primary treatment option for patients who are eligible, but only about 10-20% of patients are candidates for surgery at the time of diagnosis. For patients who are not candidates for surgery, chemotherapy is the mainstay of treatment. Colorectal cancers (CRC) rank as the third most prevalent cancers worldwide and contribute to the fourth highest number of cancer-related deaths, following lung, liver, and stomach cancers. While CRC has traditionally been more common in western countries, there has been a recent shift in its prevalence towards rapidly developing nations as well. The development of CRC can be attributed to mutations occurring in oncogenes, tumor suppressor genes, and genes associated with DNA repair. Mutations specifically in the adenomatous polyposis coli (APC) gene lead to both familial and sporadic forms of CRC. However, as APC functions as a tumor suppressor gene, the onset of CRC requires either mutations in both alleles of the gene or a mutation in one allele followed by subsequent KRAS mutation (Figure 30). Although infrequent in early adenomas, KRAS mutations can be found in up to 50% of cases once the lesion exceeds 1 cm in size. Additionally, the loss of heterozygosity or mutations in the p53 gene are closely linked to the transition from adenoma to carcinoma [START_REF] Fearon | Molecular genetics of colorectal cancer[END_REF]. In colorectal cancer, treatment options depend on the stage and location of the cancer. Surgery is the primary treatment for early-stage disease, but for more advanced cancers, chemotherapy is often used in combination with surgery or radiation therapy.

Figure 30. Schematic representation of CRC development upon occurrence of mutations. APC inactivation is responsible for the initial differentiation of the normal epithelial tissue into a dysplastic crypt. Further, mutations in KRAS lead to the formation of an adenomatous lesion, which could be followed by abnormalities in CIN, DCC, DPC4 and p53 that result in cancerous tissue in the colon. Mutations in BRAF, SMAD4 and p53 could induce metastasis [START_REF] Bender | CRISPR and KRAS: a match yet to be made[END_REF].

Lung cancer is responsible for the highest number of cancer-related deaths worldwide. Due to the difficulty of detecting lung cancer symptoms in its early stages, the diagnosis often occurs at later stages, making treatment more challenging. KRAS is frequently identified as the driver mutation in the development of lung cancer (Figure 31). However, directly targeting KRAS in lung cancer remains a significant challenge. Survival rates for lung cancer are notably lower compared to other prevalent cancer types like colorectal and prostate cancer. Patients diagnosed at earlier stages, when the tumor is confined to the lungs, have reported five-year survival rates of approximately 56%. Unfortunately, only a small percentage, around 16-17%, of lung cancer cases are diagnosed at an early stage. The five-year survival rates decline significantly to 5-6% for cases diagnosed at later stages (NIH-SEER Cancer Stats Facts: Lung and Bronchus Cancer., n.d.; [START_REF] Siegel | American Lung Association Lung Cancer Fact Sheet. CA: A Cancer Journal for Clinicians[END_REF]. NSCLC is the most prevalent type of lung cancer, and its treatment options depend on the stage and molecular characteristics of the cancer. Surgery is the preferred treatment for early-stage NSCLC, while chemotherapy and radiation therapy are used for more advanced diseases.

Figure 31. Schematic representation of driver mutations in the advancement of lung cancer. An oncogenic mutation in the KRAS gene serves as the trigger for tumorigenic progression in lung cancer, leading to the development of hyperplasia that subsequently transforms into adenoma. Mutations in the p53 and RB genes play a contributory role in the transition to a carcinogenic state within the tissue. Additionally, amplification of elements within the MAPK signaling pathway and genetic amplifications in the EGFR gene contribute to the metastasis of cancer cells to distant organs. (The intensification of cell colors signifies the progression of cancer stages.) [START_REF] Bender | CRISPR and KRAS: a match yet to be made[END_REF].

Drugging the "undruggable" KRAS

Over the last four decades, extensive efforts have been dedicated to investigating different approaches to target KRAS mutant cancers indirectly. These approaches encompass a wide range of strategies, such as modulating KRAS expression, influencing its processing and localization, targeting upstream regulatory factors, and targeting downstream effectors (Figure 32).

Figure 32. KRAS signaling pathway with main targeting inhibitors [START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

Inhibition of KRAS membrane localization

Tipifarnib and lonafarnib are both inhibitors of farnesyltransferase, an enzyme that is crucial for the prenylation and plasma membrane localization of RAS. These drugs were initially promising in mouse models and early clinical trials, as blocking KRAS localization to the membrane was thought to be an effective strategy for inhibiting downstream signaling (Figure 32) [START_REF] Adjei | A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity[END_REF]. However, despite early preclinical success, subsequent phase II and III studies failed to demonstrate clinical efficacy. This was likely due to the presence of a bypass prenylation pathway through geranylgeranylation, which limited the effectiveness of farnesyltransferase inhibitors in blocking mutant KRAS. Salirasib is a second-generation drug that inhibits the membrane localization of all activated RAS isoforms (KRAS, NRAS, and HRAS). Similarly, it did not exhibit any significant efficacy in a phase II clinical trial involving 33 patients, where none of the patients showed any response to the drug.

KRAS downstream signaling inhibition

The ineffectiveness of upstream receptor tyrosine kinases (RTKs) inhibition in the presence of activated mutant KRAS led researchers to explore downstream inhibition of the MAPK pathway to block KRAS signaling. Although there were some positive initial results of MEK inhibition either alone or in combination with chemotherapy, subsequent clinical trials showed disappointing outcomes. Both MEK inhibitors selumetinib and trametinib were tested in clinical trials for the treatment of advanced non-small cell lung cancer (NSCLC) but did not show significant benefits. Selumetinib was tested in patients who had received one or two prior lines of therapy, and no progression-free survival (PFS) benefit was observed. Similarly, trametinib was tested in patients with KRASmutant NSCLC but did not improve PFS or overall survival (OS), leading to the study being closed for futility. These findings suggest that targeting downstream MEK without any KRAS inhibition is not a feasible approach for treating KRAS-mutant NSCLC.

Preclinical studies have shown that inhibiting the downstream protein ERK can also suppress cancer cell growth and MYC degradation in KRAS-mutant pancreatic cancer cell lines [START_REF] Hayes | Longterm ERK Inhibition in KRAS-mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescencelike Growth Suppression[END_REF]. Clinical trials are underway to evaluate the effectiveness of ERK inhibitors such as ulixertinib, temuterkib, rineterkib, and JSI-1187 either alone or in combination with other agents in pancreatic, colorectal, and non-small cell lung cancers. This renewed interest in combining downstream pathway inhibition with KRAS inhibitors suggests a potential strategy for treating KRAS-mutant cancers.

It is worth mentioning that KRAS G12V is being targeted indirectly by blocking both the RAF and MEK kinases downstream. MEK inhibitors have paradoxically been found to activate RAF-induced MEK phosphorylation by inhibiting the ERK-dependent feedback loop. A dual RAF-MEK inhibitor, VS-6766, has shown promising safety and efficacy in a phase I basket trial of patients with solid malignancies or multiple myeloma that had mutations in the RAS-RAF-MEK pathway [START_REF] Lito | Disruption of CRAF-mediated MEK activation is required for effective MEK inhibition in KRAS mutant tumors[END_REF].

KRAS upstream signaling inhibition

Mutations in RAS genes make cancer cells resistant to upstream therapies that target EGFR or RTKs (Receptor tyrosine kinases). A clear example of this are the EGFR antibodies, like cetuximab and panitumumab, used only for KRAS wild-type in colorectal cancer due to this resistance. However, the development of KRAS inhibitors has led to the possibility of combining them with dual RTK or EGFR blockade. In a study of 32 patients with KRAS G12C colorectal cancer, a combination of adagrasib (MRTX849), a KRAS inhibitor, and cetuximab showed promising results with an objective response rate (ORR) of 43% and a Disease Control Rate (DCR) of 100% [START_REF] Weiss | LBA6 KRYSTAL-1: Adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation[END_REF]. The findings have prompted the initiation of a phase III clinical trial, KRYSTAL-10 (NCT04793958), which aims to compare the efficacy of second-line adagrasib and cetuximab to FOLFOX (Folinic acid, fluorouracil and oxaliplatin combination) or FOLFIRI (Folinic acid, fluorouracil and irinotecan combination) in patients with colorectal cancer carrying the KRAS G12C mutation. In a separate part of the CodeBreaK101 study, a combination of pan-ErBB inhibition with afatinib (EGFR inhibitor) and sotorasib (direct KRAS inhibitor) was administered to 33 patients with KRAS G12C-mutated cancers. The ORR was 30.3%, and the DCR was 75.8%. Nevertheless, this combination was relatively toxic, as 35% of patients experienced grade 3 or higher treatment-related adverse events (TRAEs) [START_REF] Fakih | 434P CodeBreaK 101 subprotocol H: phase Ib study evaluating combination of sotorasib (Soto), a KRASG12C inhibitor, and panitumumab (PMab), an EGFR inhibitor, in advanced KRAS p.G12C-mutated colorectal cancer (CRC)[END_REF].

RAS can be deactivated by blocking its upstream GEF, SOS1, while it switches between its active and inactive states. Several inhibitors of SOS1 which can prevent RAS from binding to GTP are being developed, including BAY-293, ARS-853, and BI-3406. These inhibitors have been shown to decrease RAS signaling in various KRAS mutant cell lines [START_REF] Hillig | Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction[END_REF][START_REF] Hofmann | BI-3406, a potent and selective SOS1:KRAS interaction inhibitor, is effective in KRAS-driven cancers through combined MEK inhibition[END_REF]. They have also been studied in combination with adagrasib and trametinib. Demonstrating high efficacy in vitro and in vivo when combined with adagrasib in KRAS G12C-mutant NSCLC models, showing superior results than adagrasib on its own. The activation of SHP2 by RTKs stimulates the interaction between GEFs and RAS, leading to the activation of the RAS-RAF-ERK pathway. Currently, several SHP2 inhibitors such as TNO155, RMC-4630, RLY-1971, and JAB-3068 are being evaluated both as single agent treatments and in combination with KRAS or other MAPK pathway inhibitors [START_REF] Mainardi | SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo letter[END_REF][START_REF] Ou | A12 The SHP2 Inhibitor RMC-4630 in Patients with KRAS-Mutant Non-Small Cell Lung Cancer: Preliminary Evaluation of a First-in-Man Phase 1 Clinical Trial[END_REF][START_REF] Ruess | Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase[END_REF]. These inhibitors hold promise in targeting SHP2 and potentially disrupting the signaling cascade involved in RAS-mediated pathways.

Direct inhibition of mutant KRAS

In the past, efforts to target KRAS mutant directly were largely unsuccessful due to two primary factors. Firstly, the RAS molecule has a strong affinity to cytoplasmic GTP, which is abundantly available in the body, making competitive inhibition difficult. Secondly, unlike other molecules targeted by inhibitors, the RAS protein has a smooth surface without a binding groove or pocket. These difficulties have led to the perception of KRAS as an undruggable target.

The laboratory of Dr. Kevan Shokat was a pioneer in the pursuit of targeting mutant KRAS [START_REF] Ostrem | K-Ras ( G12C ) inhibitors allosterically control GTP affinity and effector interactions[END_REF]. In 2013, Ostrem et al. discovered an allosteric binding site located behind the switch II region within the KRAS G12C-mutant protein. This switch II pocket was exclusive to the G12C-mutant protein that contains a cysteine residue, making it an attractive target for drug development. Indeed, cysteine is a highly reactive amino acid, and past experiments have demonstrated that active cysteine can act as a nucleophile to form covalent bonds with different agents, such as ibrutinib and dacomitinib [START_REF] Gonzales | Antitumor activity and pharmacokinetic properties ofPF-00299804, a second-generation irreversiblepan-erb Breceptor tyrosine kinase inhibitor[END_REF]. Several covalent inhibitors that attach to the cysteine in the switch-II pocket of the KRAS-GDP complex were developed. The binding of these inhibitors results in a change in shape in the switch I and switch II regions, which stops its activation and downstream signaling, eventually leading to cell death. These compounds do not interfere with the GTP-bound state and need the KRAS G12C to switch from GTP-bound to GDP-bound states through intrinsic GTPase hydrolysis for effectiveness. The introduction and eventual success of these molecules have generated great excitement in targeting what was once an 'undruggable' mutation.

KRAS G12C inhibitors

The development of specific inhibitors for KRAS G12C, such as sotorasib (AMG 510) developed by Amgen and adagrasib (MRTX849) developed by Mirati Therapeutics, had a significant and immediate impact on clinical outcomes. These small molecules are taken orally and bind selectively and irreversibly to KRAS G12C in its GDP-bound state, effectively blocking it in its inactive form (Figure 33). Initial clinical trials investigated the effectiveness of these inhibitors in various tumor types, and although positive responses were seen in different types of cancer, the use of these inhibitors alone was found to be most effective in NSCLC.

Figure 33. KRAS G12C protein structure with switch II containing adagrasib or sotorasib inhibitors [START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

The CodeBreaK100 trial investigated the use of sotorasib, a KRAS G12C inhibitor, as a monotherapy for tumors with a KRAS G12C mutation. In the trial, no dose-limiting toxicities were encountered at planned dose levels, and the recommended phase II dose was established at 960 mg. Among the 129 patients treated, 56.6% experienced treatment-related adverse events (TRAEs), but only 11.6% had grade 3 or 4 TRAEs. The response rate among patients with KRAS G12C NSCLC was 32.2%, and the disease control rate was 88.1% [START_REF] Hong | KRASG12C Inhibition with Sotorasib in Advanced Solid Tumors[END_REF].

The phase II portion of the trial explored sotorasib 960 mg once daily in cancers with a KRAS G12C mutation. The NSCLC cohort included 126 patients, 81% of whom had received prior platinum-based chemotherapy and PD(L)1 inhibitor therapy. With a median follow-up of 15.3 months, the response rate was 37.1%, and 80.6% of patients achieved disease control. The safety profile of sotorasib was consistent with that seen in the phase I portion. The most common TRAEs included diarrhea, nausea, increase in ALT and AST, and fatigue (Skoulidis et al., 2021).

The US FDA granted accelerated approval to sotorasib for patients with KRAS G12Cmutant NSCLC with at least one prior line of systemic therapy on May 28, 2021 (FDA Grants Accelerated Approval to Sotorasib for KRAS G12C Mutated NSCLC | FDA, 2021). The CodeBreaK200 trial, the first randomized phase III trial of sotorasib to report results, met its primary endpoint of progression-free survival. The study randomized 345 patients with previously treated metastatic KRAS G12C-mutant NSCLC, who had not received prior KRAS G12C inhibitors to receive sotorasib or docetaxel. The trial showed that PFS was 5.6 months with sotorasib and 4.5 months with docetaxel, and the ORR was 28.1% for sotorasib and 13.2% for docetaxel. However, the study did not meet its secondary endpoint of overall survival.

Adagrasib monotherapy for the treatment of KRAS G12C mutated non-small cell lung cancer (NSCLC), was studied in the KRYSTAL-1 phase I/II trial [START_REF] Si | First-in-Human Phase I / IB Dose-Finding Study of Adagrasib ( MRTX849 ) in Patients With abstract[END_REF]. The trial explored various doses of the drug, and the 600 mg twice daily dose was recommended for phase II trials. Among the 15 evaluable patients with KRAS G12C NSCLC treated with adagrasib 600 mg twice daily in the phase I portion of KRYSTAL-1, the ORR was 53.3%, with a median duration of response of 16.4 months and a median PFS of 11.1 months. In the phase II of the trial, 116 patients with KRAS G12C NSCLC were treated with adagrasib 600 mg twice daily, and with a median follow-up of 12.9 months, the ORR was 42.9%, the median duration of response was 8.5 months, and the median PFS was 6.5 months.

Adagrasib has also demonstrated efficacy in patients with KRAS G12C colorectal cancer, with an ORR of 22% and a disease control rate of 87% among 46 patients in a separate trial [START_REF] Weiss | LBA6 KRYSTAL-1: Adagrasib (MRTX849) as monotherapy or combined with cetuximab (Cetux) in patients (Pts) with colorectal cancer (CRC) harboring a KRASG12C mutation[END_REF]. Additionally, in a study of 33 patients with previously treated, stable central nervous system metastases, the intracranial confirmed objective response rate was 33.3% [START_REF] Jänne | Adagrasib in Non-Small-Cell Lung Cancer Harboring a KRASG12C Mutation | Enhanced Reader[END_REF]. In another report of 2 patients with untreated brain metastases, one patient had resolution of 3 untreated brain metastases and the other had a decrease in size of 3 baseline brain metastases [START_REF] Sabari | Activity of Adagrasib (MRTX849) in Brain Metastases: Preclinical Models and Clinical Data from Patients with KRASG12C-Mutant Non-Small Cell Lung Cancer[END_REF]. The drug has also shown efficacy in intracranial xenograft mouse models. Although adagrasib is associated with some dose-limiting toxicities, including nausea, diarrhea, and vomiting, it has demonstrated clinical benefit in patients with KRAS G12C mutations.

Inhibitors targeting other KRAS mutations

While new inhibitors have been approved for G12C KRAS, there is still a significant need to target KRAS mutations other than G12C, which are observed at higher frequencies. Among these mutations, G12D is the most common, followed by G12V, G12S, G12R, and others. These mutant proteins do not have an active cysteine residue and therefore require a different approach to non-covalently block the amino acid at codon 12. Another challenge in targeting these mutations, is that they lack the intrinsic hydrolysis activity observed in G12C, which suggests that they are likely to remain in the GTP-bound state [START_REF] Moore | RAS-targeted therapies: is the undruggable drugged?[END_REF].

MRTX-1133 is a noncovalent inhibitor specifically designed to target KRAS G12D. It binds to both the active and inactive states of KRAS G12D by interacting with the inhibitor binding-induced switch II pocket region (X. [START_REF] Wang | Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12DInhibitor[END_REF]. MRTX-1133 incorporates a piperazinyl group that forms an ionic bond specifically with Asp12. It has demonstrated in vivo effectiveness, inhibiting the phosphorylation of ERK and causing tumor regression in mouse xenograft models. RMC-9805 is an inhibitor specifically designed to target the active form of KRAS G12D. It achieves this by forming a unique three-component complex involving cyclophilin A, an intracellular chaperone protein. First, RMC-9805 forms a non-covalent binary complex with cyclophilin A. This binary complex then binds to the switch II pocket on the GTPbound KRAS G12D complex, resulting in the formation of a tri-complex involving KRAS, cyclophilin-A, and RMC-9805. This tri-complex leads to the formation of covalent crosslinks with the G12D mutation, effectively blocking downstream effectors from binding to KRAS. A similar approach is employed by RMC-6291 and RMC-8839, which are selective inhibitors for the KRAS G12C and G13C mutations, respectively. It should be noted that while there is a potential concern regarding the inhibition of wild-type KRAS and the associated toxicities, preclinical data indicate that the inhibition of wild-type KRAS by these inhibitors is limited.

RMC-6236 on the other hand, is a non-selective RAS inhibitor. It uses the aforementioned tri-complex formation to block interactions of multiple RAS isoforms on its active state [START_REF] Koltun | Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery[END_REF]. RMC-6236 demonstrated inhibition of all isoforms of RAS mutations, including wild type KRAS, HRAS, and NRAS, with the strongest inhibition observed in KRAS G12X-mutant cell lines. Additionally, combining RMC-6236 with an anti-PD1 immune checkpoint inhibitor showed synergistic efficacy in vivo. RMC-6236 is currently in Phase I clinical trial for solid tumor.

Mechanisms of resistance to KRAS-mutant targeting

The RAS pathways exhibit a multitude of regulatory factors both upstream and downstream, contributing to the emergence of various resistance mechanisms and alternate signaling routes that enable evasion of inhibition. Consequently, there is a potential risk that certain combination strategies may prove ineffective in combating drug resistance. Moreover, pre-clinical investigations have hinted at the existence of multiple mechanisms through which resistance can develop, encompassing innate, acquired, and adaptive tumor responses that diminish the therapeutic effectiveness of KRAS mutation inhibitors. The subsequent section will focus on the specific mechanisms underlying resistance to inhibitors targeting KRAS mutations.

Resistance to KRAS G12C inhibitors

The efficacy of adagrasib and sotorasib in treating KRAS-mutant cancers is affected by factors such as concurrent mutations, smoking history, and tumor microenvironment. In the CodeBreaK100 and KRYSTAL-1 trials, less than 50% of patients achieved any objective response, indicating innate resistance to treatment. Concurrent mutations can also be associated with innate resistance to KRAS inhibition (Figure 34). Moreover, concurrent mutations have been associated with innate resistance to KRAS inhibition. Mutations in the tumor suppressor genes STK11 (serine/threonine kinase 11) and KEAP1 (Kelch-like ECH-associated protein 1), have been found to have a negative prognostic and potentially predictive function.

Furthermore, a primary resistance may occur due to a diverse reaction, where certain cells are eradicated by the kinase inhibitor, while others produce new KRAS G12C that ultimately transforms into an active form that is resistant to the medication. Epithelialto-mesenchymal transition (EMT) is another mechanism of innate and acquired resistance to KRAS inhibition. These findings suggest a need to better understand the mechanisms of innate resistance to further tailor molecular treatments for KRAS-mutant cancers.

Figure 34. Resistance mechanisms to sotorasib and adagrasib [START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

Efforts are being made to comprehend the acquired resistance to selective KRAS G12C inhibitors. There are four broad categories of acquired resistance mechanisms: (1) ontarget resistance, which includes secondary or concurrent KRAS alterations, (2) off-target resistance, such as bypass mechanisms upstream, downstream or parallel to KRAS, (3) changes in the tumor microenvironment (TME) and (4) histological transformation (Figure 34). Various resistance mechanisms have been identified, including concurrent KRAS alterations, changes in the microenvironment, vertical signaling changes such as RTK-RAS-MAPK, and phenotypic transformation. In some cases, multiple resistance mechanisms occur simultaneously.

Other mutations in addition to KRAS G12C can occur simultaneously in KRAS-driven cancers. A combined analysis of multiple cancers showed that 2.8% of cases have multiple KRAS mutations. A recent study of a large group of patients with KRAS NSCLC found that 8% of them had an additional KRAS mutation, which could result in either KRAS G12F or KRAS G12V proteins depending on the location of the mutation [START_REF] Vaclova | Concomitant KRAS mutations attenuate sensitivity of non-small cell lung cancer cells to KRAS G12C inhibition[END_REF]. In vitro studies have shown that cells with both KRAS G12C and G12V mutations are resistant to KRAS G12C inhibition. Resistance can be acquired due to the activation of non-G12C KRAS mutations, including G12D/V/R, G13D, and Q61H, which are often in a trans allelic configuration. Another type of resistance can be triggered by an allelic rearrangement of G12C to G12W. The on-target KRAS activating mutations of switch-II binding pocket, involving KRAS Y96, R68 and H95 residues has been found to provide resistance to allele-specific KRAS G12C inhibitors [START_REF] Awad | Acquired Resistance to KRAS G12C Inhibition in Cancer[END_REF]. Additionally, G12C inhibitors have been observed to cause an increase in MAPK signaling by upregulating and phosphorylating RTKs such as EGFR, HER2, FGFR, ALK, and MET, due to reactivated HRAS and NRAS signaling [START_REF] Ryan | Vertical pathway inhibition overcomes adaptive feedback resistance to KRASG12C inhibition[END_REF]. Studies have shown that inhibiting upstream RTKs has been effective in restoring sensitivity to sotorasib in NSCLC, both in vitro and in vivo.

Precise targeting of KRAS using CRISPR-cas Systems

The CRISPR systems offer significant opportunities for targeting mutations due to their ease of use in genome editing. In recent years, researchers have focused on diseasecausing mutations, particularly in diseases without current cures like cancer. Although limited, the existing literature on CRISPR applications targeting KRAS mutations has shown promising results. While molecules in the KRAS pathway have been more frequently targeted using CRISPR than KRAS itself, it is evident that the full potential of CRISPR for targeting KRAS mutations in cancer has yet to be fully realized.

Direct targeting of mutant KRAS variants using CRISPR systems has led to a significant reduction in cell viability and proliferation in laboratory settings, as well as inhibition of tumor growth in animal models. Furthermore, knockdown of mutant KRAS through CRISPR has been observed to regulate downstream molecules such as PI3K, ERK, AKT, Stat3, and c-myc.

The effectiveness of the CRISPR system in suppressing KRAS function has been evaluated targeting the most frequently mutated KRAS isoforms in cancer cells and animal models, as presented in Table 4.

CRISPR system

Targeted molecules

Cancer type

Delivery method

Delivery mode

Cell type Reference

CRISPR-Cas9

Whole

CRISPR applications for direct targeting of G12D and G12V KRAS mutations

Several studies have explored different approaches to understanding and targeting KRAS mutations in pancreatic and colorectal cancers. One notable study by Lee et al. in 2018 used lentivirus to deliver the CRISPR-Cas9 system in its DNA form and introduce indels and induce frameshift mutations at the KRAS codon-12 sites in pancreatic and colorectal cancer cells (W. Lee et al., 2018). Specifically, they targeted KRAS c.35G>T and c.35G>A, resulting in G12V and G12D mutants, respectively. The disruption of cell proliferation was observed in cancer cells with the specific KRAS mutations, while cells with wild-type KRAS were unaffected. The researchers evaluated cell viability using an MTT proliferation assay and observed a significant decrease in cell numbers following transduction with sgRNA targeting KRAS G12V. However, the transduction of SNU407 and AS-PC1 cells with sgRNA targeting KRAS G12D did not negatively impact cell viability, likely due to low indel rates associated with cell line-specific transduction efficiency.

Another important study conducted by Kim et al., investigated the targeting of these frequently mutated KRAS forms using lentivirus to deliver CRISPR-Cas9 DNA form in various cancer cell lines and assessed its impact on cell proliferation, survival, and cancer growth both in vitro and in vivo (W. Kim et al., 2018). They successfully achieved significant downregulation of mutant KRAS variants, including G12V, G12D, and G13D, while leaving the wild-type KRAS unaffected. The survival and tumorigenicity of KRAS mutant cells were significantly inhibited, whereas cells with wild-type KRAS were not affected. By disrupting mutant KRAS using Cas9 and sgRNA, a remarkable reduction in tumor size (7-fold to 2-fold) was observed in KRAS mutant mice. The researchers concluded that the combination of CRISPR-Cas9 with existing cancer therapies could enhance their effectiveness. However, they cautioned against using CRISPR-Cas9 as a monotherapy due to its less than 100% efficiency and the potential risk of causing additional mutations.

These studies contributed to the understanding of the potential therapeutic strategies for targeting G12D and G12V main KRAS mutations using CRISPR-Cas9, but further research is needed for practical application.

Aims of the project

Genome editing mediated by CRISPR-Cas9 has shown promise for the treatment of point mutation diseases. This gene editing tool offers a wide range of therapeutic possibilities for treating cancer, if suitable delivery vectors are developed. Currently, adenoassociated viruses are the most widely used vectors for CRISPR-Cas9 gene therapies. However, limitations such as packaging capacity, long-term Cas9 expression, and potential immune responses raise concerns about their clinical applicability in gene editing strategies. Peptide-based nanoparticles (PBN) mediated delivery of CRISPR-Cas9 components could offer a safer alternative for delivering gene editing reagents systemically.

Successful delivery of Cas9 protein and its gRNA as ribonucleoprotein (RNP) complexes by PBN has been reported. However, the feasibility of co-delivering Cas9 mRNA and its sgRNA has not been reported thus far.

The objective of my PhD project was to investigate the feasibility of using novel ADGN peptide-based nanoparticles, developed by Divincell company, to deliver efficiently CRISPR-Cas9 mRNA and gRNA targeting specific genes in vitro and in vivo (Figure 35).

The first part of my PhD project focused on optimizing the physicochemical properties of ADGN-PBN, including surface charge, size, and homogeneity, to ensure effective encapsulation and protection of large RNA cargoes. ADGN-PBN cellular internalization and RNA cargo release were also analyzed.

A second part sought to test the functionality of the delivered CRISPR-Cas9 system to target and edit the luciferase reporter gene expressed in human cancer cells. The project aimed to achieve high editing efficiency, to validate the knockout of the target gene using different assay methods, and to analyze the mutations induced by the CRISPR-Cas9 system on the target DNA.

Lastly, the potential of ADGN-PBN as a safe and effective delivery system for CRISPRbased gene editing, particularly targeting the challenging KRAS cancer mutations, was aimed to be demonstrated.

Results part 1: ADGN-PBN are an efficient mRNA delivery system

Cell penetrating peptides (CPP) have been described as one of the most efficient vectors for achieving cellular uptake of different molecules, including siRNA and more recently, small mRNA cargoes (around 1000 nucleotides). The first goal of my project was to evaluate the potential of ADGN peptides to serve as mRNA nanocarriers for cancer cells transfection. This is a key point of the project because the CRISPR-Cas9 system will be administered in its RNA form and Cas9 mRNA is a particularly large molecule (4521 nucleotides). To evaluate whether ADGN peptides are a good candidate for mRNA delivery, we first assessed its complexation capacity with mRNA molecules and its ability to form stable non-covalent peptide-based nanoparticles. Following, the transfection efficacy on different cancer cells was evaluated in terms of protein translation.

Innovative ADGN peptides show interesting properties for RNA delivery

CPPs have been reported as highly effective carriers for facilitating the internalization of various cargoes into cells, including siRNA molecules [START_REF] Multiplex Crombez | A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells[END_REF]. Nonetheless, their use in cancer therapy is significantly restricted because they lack tissue selectivity, distributing widely across most tissues upon in vivo administration. To address this limitation and enhance tumor-specific delivery, researchers have developed a combination comprising the cell-penetrating peptide CADY and a targeting ligand called the laminin receptor binding peptide (YIGSR) [START_REF] Laroui | Improvement of Cell Penetrating Peptide for Efficient siRNA Targeting of Tumor Xenografts in Zebrafish Embryos[END_REF]. This combination aims to target the 37/67 kDa laminin receptor, which is commonly upregulated in cancer cells and associated with their invasiveness and metastatic potential. The resultant targeted CPP, named "Y-CADY," did not alter CADY's physico-chemical properties and demonstrated superior efficacy compared to CADY in gene silencing within cancer cell lines and exhibits increased potency in targeting and inducing apoptosis in tumor xenografts in zebrafish embryos. Even though CADY and Y-CADY have showed great promise as siRNA delivery vehicles, its complexation with bigger RNA molecules, as messenger RNA, is weaker and less stable, affecting considerably its delivering efficiency.

On the basis of CADY and Y-CADY sequence as a template, we engineered two secondary amphipathic peptides: ADGN-ava [Ac-YIGSR-Ava-LWRALWRLLRSLWRLLWK-NH2] and ADGN-peg2 [Ac-PEG2-LWRALWRLLRSLWRLLWKR-NH2] with different properties that may spontaneously create non-covalent complexes with big RNA molecules. Indeed, similar to CADY peptide, ADGN peptides may interact with nucleic acid molecules creating nanoparticles with a cage-like structure or nanocages [START_REF] Crowet | Modeling of non-covalent complexes of the cell-penetrating peptide CADY and its siRNA cargo[END_REF]. ADGN peptides self-assemble with the nucleic acids mainly through electrostatic interactions between the cationic arginine residues and the mRNA phosphate backbone.

ADGN peptides can adopt multiple positions at the RNA surface due to the different positions of the arginine residues on the peptides and the numerous phosphate moieties in the RNA structure. In addition, the presence of tryptophane residues in ADGN sequence improve its ability to interact with the lipid phase of the membrane [START_REF] Jobin | The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides[END_REF]. This leads to numerous possibilities for ADGN complexation, while maintaining several common complex properties as the average size distribution and surface charge.

Moreover, the three-dimensional models of ADGN peptides was model based on circular dichroism (CD) measurements. Results showed that ADGN peptides folded into an helical structure upon interaction with RNA molecules while staying unfolded with random coil CD spectra in its free form. Figure 36 exhibit the visualization of the adequate helical conformation of ADGN peptides, exposing of tryptophan aromatic residues on one side, charged residues on the other and hydrophobic residues on yet another to facilitate RNA interaction. In contrast to CADY peptide that was synthesised with a cysteamide group (cya) at the C terminus, ADGN peptides contain an amide group (NH2) that mimics more closely the native proteins and therefore may increase the biological activity of the peptide while increasing its stability.

Similar to Y-CADY, ADGN-ava contains a targeting motive, the laminin receptor binding peptide (YIGSR). YIGSR interacts with the 37/67 kDa laminin receptor, which is known to be overexpressed in a majority of cancer cells and is associated with their invasive and metastatic characteristics. By binding to this receptor, the YIGSR peptide facilitates the internalization of the cargo molecules [START_REF] Laroui | Improvement of Cell Penetrating Peptide for Efficient siRNA Targeting of Tumor Xenografts in Zebrafish Embryos[END_REF][START_REF] Meng | Enhanced gene transfection efficiency by use of peptide vectors containing laminin receptor-targeting sequence YIGSR[END_REF]. In contrast to Y-CADY, ADGN-ava substitutes the serine (S) linker and glycine (G) residue with 5aminovaleric acid (ava) linker which provides higher flexibility to the two amino acid domains (Table 5 and Figure 36).

ADGN-peg2 was covalently attached to a polyethylene glycol molecule (PEG2) which is a nontoxic, biocompatible polymer that enhances complex stability, prolongs half-life through reduced renal clearance and masks antigenicity for minimum immune response (Figure 37) [START_REF] Suk | PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[END_REF]. The combination of ADGN-ava and ADGN-peg2 peptides in a 60:40 weight ratio will be referred to as "ADGN" peptides in this document and was used to provide both the targeting motive from ADGN-ava and the stability contributed by ADGN-peg2 to the complex.

Peptide Sequence

CADY Ac-GLWRALWRLLRSLWRLLWRA-cya Y-CADY YIGSRSGLWRALWRLLRSLWRLLWK ADGN-ava Ac-YIGSR-Ava-LWRALWRLLRSLWRLLWK-NH2 ADGN-peg2
Ac-PEG2-LWRALWRLLRSLWRLLWKR-NH2

ADGN peptides effectively complex with mRNA molecules

The ability of these new ADGN peptides to interact with larger RNA molecules was determined. Indeed, the delivery of mRNA molecules implies a more difficult technical challenge due to the bigger size (more than 100 kDa) of the mRNA molecules compared to the previously described cell-penetrating-peptide delivery of siRNA molecules whose size range between 20 and 25 nucleotides (4-14kDa). This is particularly important as one of my thesis goals is to vectorize the large full length Cas9 mRNA.

Foremost, we decided to use mRNA which encodes for luciferase protein (fLuc), originally isolated from the firefly Phothinus pyralis and that is composed of 1929 nucleotides in length corresponding to 638 kDa. The ratio between negatively charged phosphorous in the mRNA molecule and the positively charged residues from the peptides is decisive for the mRNA encapsulation efficacy, physicochemical nanoparticle characteristics and biological activity. We therefore assessed the nucleic acid encapsulation capability of the ADGN peptide-based nanoparticles (ADGN-PBN) at different peptide to RNA weight ratios by electrophoretic mobility shift assay (Figure 38A). Results revealed an increase on percentage of RNA complexed within the ADGN-PBN with increasing peptide to RNA ratios. A maximal encapsulation efficiency was reached with ratios higher than 2:1 (Figure 38B). Confirming that the amphipathic conformational structure of the ADGN peptides allowed its complexation with big mRNA molecules and could potentially be used as its delivery system.

ADGN-PBN show a monodisperse size distribution

Nanoparticle size distribution plays a critical role when used as delivery systems, especially for systemic delivery as it directly affects the pharmacokinetics and the efficacy of the cargo. When nanoparticles are administered systemically, they are subject to various biological barriers, such as the reticuloendothelial system (RES), which can recognize and clear foreign particles from the bloodstream. Nanoparticles with larger sizes tend to be rapidly detected and cleared by the RES leading to shorter circulation time and lower bioavailability. On the other hand, nanoparticles that are too small may be rapidly eliminated by renal clearance thereby limiting their potential to accumulate in target tissues. Therefore, it is essential to optimize the size distribution of ADGN-PBN in order to achieve the desired therapeutic effect. This can be achieved by controlling the conditions during the complexation process. The constant agitation during nanoparticles assembly, the speed of agitation, the temperature of the reagents, the slow and dropwise addition of the peptide as well as the concentration of the reagents are critical variables during nanoparticle size optimization (Table 6).

Table 6. Conditions tested for optimal complexation of ADGN peptides with mRNA molecules.

After optimization of nanoparticle complexation conditions to a constant agitation at 400 rpm, pre-warming of the solutions at 37°C, addition of the reagents at a medium dropwise speed (5 seconds between each drop) and a total volume of 600 µl, we analyzed the size distribution of ADGN-PBN complexed with fLuc mRNA by dynamic light scattering. 0.8 µg of fLuc mRNA were complexed with 1.6 µg of ADGN peptides to form peptide based nanoparticles with a 2:1 peptide:RNA weight ratio. ADGN-PBN were able to form monodispersed complexes of 91.87 ± 1.5 nm in size (Figure 39), a slightly negative surface zeta potential of -9.81 ± 1.5 mV and a polydispersity index of 0.17 ± 0.02 (Table 7 and Figure 39). ADGN-PBN are therefore within the size range that has been shown to have certain advantages as improved circulation times and increased accumulation in tumor tissues due to the enhanced permeability and retention (EPR) effect. Even though the neutral or negatively charged nanoparticles are taken up less efficiently than positive nanoparticles, ADGN-PBN would rely on other factors influencing cellular uptake such as the particle size, surface functionalization and shape.

Peptide to RNA ratio highly influence transfection efficiency

In addition to the effect on the physico-chemical properties of the nanoparticles, the peptide to RNA ratio can significantly affect the biological response induced on living cells. To evaluate this, pancreatic cancer cells (Panc-1 cells) were transfected with ADGN-PBN complexed with 0.8 µg of fLuc mRNA at increasing peptide to RNA ratios (2:1, 3:1, 5:1 and 10:1). The luciferase expression levels of the cells were evaluated 48 hours posttransfection using the luciferase assay system from Promega. The cells reached a maximum luciferase expression level after transfection with ADGN-PBN with a 2:1 peptide:RNA weight ratio and it progressively decreased with increasing ratios (Figure 40). These results confirmed the importance of an optimal peptide to RNA ratio to obtain the desired biological effect.

ADGN-PBN induced luciferase expression on cancer cells

After the selection of an optimal peptide to RNA ratio based on the physico-chemical characteristics of the nanoparticles and transfection efficiency on Panc-1 cells, its transfection efficiency was evaluated on human lung cancer cell lines (H441, H358 and A549) and epithelial adenocarcinoma cells (Hela). The levels of luciferase expression were assessed after cellular transfection of ADGN-PBN complexed with 0.8 µg of fLuc mRNA at 2:1 peptide:RNA weight ratio.

These parameters are the best ones tested in the previous experiments. Luciferase expression levels were measured 48 hours after transfection. Results showed that all cell lines had a statistically significant increase of luciferase activity after transfection with fLuc mRNA complexed with ADGN-PBN (Figure 41). However, the performance of ADGN-PBN as transfection agent to deliver mRNA cargo strongly depends on the cell type, reaching higher efficiencies in Hela and A549 cells. Luciferase activity was 2 to 3 times less efficient in H441, H358 and Panc-1 cell lines. These variations in transfection efficiency may be explained by the fact that these cell lines do not have the same characteristics. Indeed, it was demonstrated that some of the cell dependent factors like the proliferation rate of the cells, the cellular metabolism, the presence of specific cell surface receptors and the properties of the cell membrane, such as its fluidity, lipid composition and thickness can affect the cellular uptake of nanoparticles [START_REF] Neuhaus | Nanoparticles as transfection agents: A comprehensive study with ten different cell lines[END_REF]. Cell penetrating peptides have been used as delivery vectors for small RNA molecules.

The secondary amphipathic structure of ADGN cell penetrating peptides favors its interaction with bigger mRNA molecules, forming stable non-covalent nanoparticles. These ADGN-PBN are able to deliver the mRNA cargo into cancer cells reaching high transfection efficiencies depending on the peptide to RNA ratio and the cell type. ADGN-PBN are therefore a promising delivery system for mRNA therapeutics. The next step of the project was to evaluate ADGN-PBN capacity to deliver the larger RNA components of the CRISPR-Cas9 system to cancer cells.

Results part 2: ADGN-PBN as a delivery system for CRISPR-Cas9

The second part of my work consisted in the evaluation of the capability of ADGN peptides to deliver a functional CRISPR-Cas9 system. We already proved that ADGN-PBN are able to deliver mRNA molecules of around 2000 nucleotides. However, one of the major challenges associated with the CRISPR-Cas9 system is the large size of the Cas9 mRNA (4521 nucleotides and 1500 kDa). In addition, ADGN peptides should interact with a second RNA molecule corresponding to the sgRNA (100 nucleotides and 34 kDa). In order to validate the functionality of the CRISPR-Cas9 components after transfection mediated by ADGN-PBN, we proceeded to: first, verify the capability of ADGN peptides to complex with the RNA components of the CRISPR-Cas9 system and to form stable nanoparticles; second to corroborate the cellular uptake of CRISPR-Cas9 mediated by ADGN-PBN; third to evaluate the gene editing efficiency of the CRISPR-Cas9 system after delivery with ADGN-PBN; and finally, to investigate the possible mutations induced by the system.

ADGN peptides complex with the CRISPR-Cas9 components forming stable PBN

In order to determine the capacity of the ADGN peptides (ADGN-ava and ADGN-peg2) to interact with the CRISPR-Cas9 RNA components and to form stable nanocomplexes, we used the electrophoretic mobility shift assay. This test was evaluated at various peptide:RNA weight ratios (0.25:1, 0.5:1, 1:1 and 2:1) (Figure 42A). The results show that ADGN-PBN have high capacity of complexation with CRISPR-Cas9 RNA components. Cas9 mRNA and sgRNA were stably encapsulated into the nanoparticles at peptide:RNA weight ratios higher of 1:1. The quantification of the detected RNA fragments showed that optimal complexation between ADGN peptides, Cas9 mRNA and sgRNA occurred at a 1:1 peptide:RNA (Cas9 mRNA + sgRNA) weight ratio with a complete encapsulation with a 2:1 weight ratio, where no free RNA fragments were detected (Figure 42B). As mentioned before, the proper size distribution and zeta-potential of this new nano delivery system are important parameters for transfection efficiency. We analyzed then the size distribution of each ADGN-PBNs containing CRISPR-Cas9 RNA components at different weight ratios (0.5:1, 1:1 and 2:1) via dynamic light scattering (DLS). The average hydrodynamic diameter of the three types of ADGN-PBNs ranged from 109,57 to 139,63 nm with a polydispersity index (PDI) between 0.18 and 0.22 indicating a monodisperse distribution and the absence of aggregates. The proper assembly of nanoparticles was also confirmed by the zeta potential (Table 8), which was negative at 0.5:1 peptide:RNA weight ratio (-12,6 mV) and became positive (7.49 mV) with the increase of the peptide:RNA weight ratio. This result is in adequation with the complete RNA complexation with the ADGN peptide observed on the gel retardation assay at the peptide:RNA weight ratio 2:1 (Figure 42). The morphology of the ADGN-ava Cas9 mRNA/sgRNA complex as well as the particle size was characterized by transmission electron microscopy (TEM). PBN nanoparticles at 2:1 peptide:mRNA weight ratio exhibit a spherical shape structure with an average homogenous size distribution between 80 and 120 nm as determined by DLS (Figure 43A) and they are well dispersed as individual nanosystems (Figure 43B). These results confirmed the ability of ADGN peptides to form stable PBN with the CRISPR-Cas9 RNA components that are suitable for cellular delivery.

RNA cargo is protected from nuclease degradation by ADGN-PBN

As mentioned before, RNA is inherently unstable to degradation by nucleases that are presence in the extracellular environment. If the RNAs are degraded, they will not be able to form the CRISPR-Cas9 machinery and direct the gene editing. Thus, nuclease protection is critically important when nanoparticles are used for the delivery of CRISPR-Cas9 RNA components. We further hypothesized that the formation of a such peptide nanocage around the RNA cargo might prevent its degradation by RNAse. Consequently, the RNase-protective effect of nanocage forming ADGN-PBN was evaluated using gel electrophoresis. In this assay, free RNA or RNA complexed in ADGN-PBN was incubated in the presence or absence or RNAse A. To release the RNA from the peptide nanocage, proteinase K was used to dissociate RNA from the ADGN-PBN and facilitate RNA integrity analysis. RNAsecure was used to inactivate RNAse A before rupture of the nanoparticles. As depicted in Figure 44, after incubation with RNAse A at 10 ng/mL, naked RNA was subject to degradation by RNase A. In contrast, when RNA is encapsulated into ADGN-PBN incubated with increasing concentrations of RNAse A (10-100 ng/mL) it is fully protected from degradation. As expected, the addition of RNAse inhibitor (RNAsecure) inhibits the action of RNAse A and maintains RNA integrity after dissociation from the ADGN-PBN complex. This result demonstrated that the peptide nanocage formed by ADGN-PBN can effectively protect the RNA cargo from RNAse A that is present in external environment. Thus, increasing the likelihood that the RNAs will successfully reach the intended target, form the CRISPR-Cas9 machinery and effectively edit the gene of interest in cells.

Figure 44. Efficient RNA protection by ADGN-ava from RNAse A degradation. 0.5 μg of Cas9 mRNA was complexed with 1 μg of ADGN-ava peptide to form peptide based nanocages that were digested with 0, 10, 50, 100 and 500 ng/ml of RNAse A at 37°C for 30 minutes. After digestion, RNAse was degraded by RNAsecure and subsequently the ADGN-PBN were digested with proteinase K to break the complex and remove the peptide. Remaining solution was electrophoresed on 0.8 % agarose gel and stained with GelGreen nucleic acid stain. Released RNA is visualized as black bands on the gel.

ADGN-PBN mediate cellular uptake of CRISPR-Cas9 components

In vitro RNA transfection mediated by this novel ADGN-PBN was firstly investigated using cyanin 3 (Cy3) fluorescently labelled sgRNA and cyanin 5 (Cy5) fluorescently labelled ADGN peptide. A549 cells were used as ADGN-PBN previously showed high transfection efficiencies on this cell line. The cellular uptake of the complexes was evaluated by fluorescence activated cell sorter (FACS) after 3 hours incubation with labelled ADGN-PBN with a peptide to RNA weight ratio of 1:1. The flow cytometry data in Figure 45 showed that after transfection with ADGN peptide complexed with Cy3-sgRNA and nonlabelled mRNA, 99.3% of the cells were cyanine 3 positive, compared to only 30% positive cells after incubation with free sgRNA-Cy5, suggesting that ADGN peptide mediated efficiently the cellular interaction of the RNA cargo. The percentage of positive cells in absence of nanoparticle carrier may be explained by the capacity of small RNA molecules as siRNA and miRNA to diffuse across the cell membrane [START_REF] Duxbury | Systemic siRNA-mediated gene silencing: A new approach to targeted therapy of cancer[END_REF][START_REF] Glud | Naked siLNAmediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration[END_REF]. It has to be noted that the transfection capacity of naked RNA molecules is typically low and should be improved by specialized delivery systems for therapeutic purposes. The percentage of fluorescent cells was also high when cells were transfected with Cy5-ADGN peptide complexed with non-labelled mRNA and non-labelled sgRNA implying high interaction of the Cy5-ADGN peptide with the cells. Similarly, 92.5% of the cells were double positive when transfected with Cy5-ADGN peptide complexed with Cy3-sgRNA and Cas9 mRNA, suggesting that the nanoparticles are tightly interacting with the cells. Interestingly, the Cy3 fluorescence intensity of this double positive cell population (transfected with Cy5-ADGN peptide complexed with Cy3-sgRNA and Cas9 mRNA) was lower than the Cy3 fluorescence intensity of the cells transfected with ADGN peptide complexed with Cy3-sgRNA and non-labelled mRNA, suggesting a possible quenching effect of the Cy5-labelled peptide. The cellular uptake kinetic was further analyzed using spinning disk confocal microscopy after the incubation of A549 cells with ADGN-PBN consisting of Cy5-labelled ADGN-ava

ADGN peptides closely interact with the RNA cargo

To validate our hypothesis that the lower cyanin 3 fluorescence intensity observed by confocal microscopy and flow cytometry was due to the close complexation with the Cy5 labelled peptide that may induce an energy transfer between the fluorophores, we performed a fluorescence resonance energy transfer (FRET) assay. FRET is a distancedependent energy transfer process between two fluorophores, one of which acts as an energy donor fluorophore and the other as an acceptor. Thus, FRET can be used to estimate the nanoscale distance between two interacting biomolecules. The proximity between the donor and acceptor should be <10 nm. This highly sensitive technique has been widely used as a powerful tool to study the intermolecular distance between fluorophores and molecular conformational changes. An important condition required for FRET to occur is the overlap of the donor emission spectrum with the acceptor absorption spectrum. Fluorescence spectrum of labelled ADGN-PBN complexed with CRISPR-Cas9 at different peptide to RNA weight ratios (1:1 and 0.5:1) were analyzed after excitation at 496nm to detect a possible FRET effect. The complex was formed with ADGN-ava peptide labelled with the acceptor fluorophore cyanin 5, mRNA Cas9 and sgRNA labelled with the donor molecule cyanin 3. Fluorescence spectrums are represented on Figure 48. Results showed an increase in the fluorescence emission of ADGN-PBN labelled with Cy3 and Cy5 at the acceptor (Cy5) emission wavelengths at both 0.5:1 and 1:1 ratios (light and dark blue) compared to ADGN-PBN only labelled with the Cy5 molecule (red). Moreover, we observe a decrease in the fluorescence intensity at the donor Cy3 emission range when higher peptide to RNA ratio is used (higher presence of acceptor Cy5-ADGN peptide), suggesting higher energy transfer.

Table 9 shows the fluorescence intensity values at 565 nm and 685 nm of ADGN-PBN formed with Cy5-labelled peptide and/or Cy3-labelled sgRNA and mRNA Cas9 non labelled at different peptide to RNA weight ratios, after excitation at 496 nm. The fluorescence intensity of the acceptor Cy5 coupled to ADGN peptide increases after excitation at 496 nm (wavelength that corresponds only to the excitation of Cy3 molecule coupled to sgRNA) when it is closely complexed with Cy3-labelled sgRNA. Meanwhile, the fluorescence intensity of Cy3-labelled sgRNA decreases in presence of the acceptor Cy5-ADGN peptide. FRET efficiency calculated by the acceptor photobleaching method is 87% and 66% for ADGN-PBN at 1:1 and 0.5:1 respectively. These results are in accordance with the lower Cy3 signal observed 1 hour after ADGN-PBN transfection by confocal microscopy as the two fluorophores are still in close physical proximity (Figure 46). After 2 hours, when the complexes start to dissociate, the Cy3 fluorescence intensity is higher, and less energy transfer could occur. ADGN peptides form stable non-covalent peptide-based nanoparticles with the RNA components of the CRISPR-Cas9 system. ADGN-PBN complexed with CRISPR-Cas9 have a particle size distribution of around 100nm with a low polydispersity index. The cage like structure that surrounds the CRISPR-Cas9 RNA components protects the system from nuclease-mediated degradation. Furthermore, ADGN-PBN promoted cellular uptake of the system and delivered it intracellularly, allowing the further translation of the Cas9 protein for the assembly of the CRISPR-Cas9 machinery. It is worth noting that conducting additional analyses such as fluorescence lifetime imaging microscopy (FLIM) would provide a more comprehensive investigation into the potential energy transfer between the labeled peptide and labeled RNA, both prior to and after cellular internalization and RNA release.

ADGN-PBN mediated CRISPR-Cas9 delivery into mammalian cells induces knock out of an exogenous gene

ADGN-PBN significantly decrease luciferase expression

Once the CRISPR-Cas9 RNA cargo is delivered to the cells, the next step is to validate whether these RNAs are still functional and therefore if Cas9 mRNA is able to translate in Cas9 protein that recognizes the sgRNA to form the CRISPR-Cas9 machinery and to perform gene editing. To assess the knock-out efficiency mediated by CRISPR-Cas9, after the delivery by ADGN-PBN, the exogenous gene luciferase was targeted. A549-luc and MCF7-luc cells stably expressing luciferase were transfected with ADGN-PBN complexed with Cas9 mRNA and sgRNA targeting luciferase. The luciferase expression was measured 48 hours after transfection. A peptide to RNA weight ratio of 1:1 was used as it previously showed optimal physico-chemical properties for transfection, high CRISPR-Cas9 encapsulation efficiency and RNAse protection. In parallel, experiments with the well-known commercially available polyplus JetMessenger transfection agent were performed for comparison. In A549-luc cells, ADGN-PBN peptide complexed with 0.5 µg Cas9 mRNA and 0.5 µg sgRNA with a 1:1 peptide to RNA weight ratio, induces a 50% decrease in luciferase activity that is comparable with polyplus JetMessenger transfection agent whereas 5 time less RNA is used in this case. The ADGN-PBN complexed with CRISPR-Cas9 RNA efficiently inhibit luciferase activity in a RNA dose-dependent manner (Figure 49). In contrast, no change in luciferase activity was observed after the transfection of ADGN-PBN containing a non-targeting control sgRNA showing that luciferase activity decrease was attributable to the selected sgRNA and not to the vector. In MCF7-luc cells, similar results were obtained with a 60% decrease in luciferase activity for the same delivered RNA quantity. It should be noted that polyplus JetMessenger transfection did not induce a significant decrease in luciferase activity in this cell line, which could be attributed to cell dependent factors mentioned before.

An important criterion for the development of a new CRISPR-Cas9 delivery carrier is its safety and cellular non-toxicity. As depicted in Figure 49 C and D, no toxicity or alteration of cell viability related to the use of CRISPR-Cas9 delivering ADGN-PBN complex was detected for all the parameters tested using an MTS assay. Overall, these results demonstrate that optimization of RNA quantity delivered by ADGN-PBN resulted in efficient and specific gene silencing by CRISPR-Cas9 system. compared after transfection of CRISPR-Cas9 system targeting luciferase. 0.25 or 0.5 ug of Cas9 mRNA and 0.5 ug of sgRNA targeting luciferase were encapsulated in ADGN-PBN with a 1:1 peptide:RNA weight ratio or in polyplus JetMessenger transfection agent (mean ± sem, n = 3 biologically independent samples). *Indicates p < 0.05 between groups. **Indicates p < 0.01 vs. control vector unless otherwise labeled. Statistical analysis was performed using a one-tailed non-parametric t test. A549-luc (C) and MCF7-luc (D) cell viability were determined for each conditions mentioned above using MTS test.

ADGN-PBN induce mismatched heteroduplex formation

The editing efficiency of CRISPR-Cas9 delivered by ADGN-PBN was evaluated at the DNA level by the indirect method called T7E1 mismatch detection assay. This assay relies on the ability of the T7 endonuclease I (T7E1) to recognize and cleavage heteroduplex DNA molecules that contain mismatches between the edited and unedited DNA strands. A549luc cells were transfected with ADGN-PBN complexed with 0.5 µg mRNA and 0.5 µg sgRNA with a peptide:RNA weight ratio of 1:1. Total DNA was extracted 48 hours after transfection, PCR amplified, denatured and reannealed to form DNA heteroduplexes that where finally cleaved by the T7 endonuclease I (Figure 50A). Fragments were then analysed by agarose gel electrophoresis.

Heteroduplex formed by edited and non-edited sequences have conformational changes that make them migrate significantly slower during gel electrophoresis compared to homoduplexes of two non-edited or two edited sequences and therefore they can be distinguished in the absence of the endonuclease.

Cells transfected with polyplus JetMessenger transfection agent reagent were used for comparison. The T7E1 results demonstrated that the cleavage efficiency in cells transfected with ADGN-PBN complexed with CRISPR-Cas9 system targeting luciferase was 60% compared to no cleavage for non-treated A549-luc cells (Figure 50B).

ADGN-PBN induce luciferase gene knock-out

Despite the convenience of mismatches detection methods, they are only semiquantitative as the number of heteroduplex is not directly measuring the percentage of indels. To overcome these disadvantages, a quantitative PCR based mutation detection method was also used. This allows to not only detect but also quantify the percentage on indels caused by non-homologous end joining repair after CRISPR-Cas9 treatment. Via the amplification of the respective target locus [START_REF] Germini | A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools[END_REF]. A first primer is designed to bind near the predicted mutation site (primers OUT) whereas a second primer is design to bind to the wild type sequence of the predicted mutation site (primers IN) (Figure 51A). As the primers are highly specific for the wild type sequence, the mutations induced by CRISPR-Cas9 impairs the amplification. Thus, the gene editing efficiency can be measured as a decrease in the qPCR signal relative to the control sample amplified with primers outside of the expected mutation site (primers OUT).

As shown in Figure 51B, the relative fluorescent signal significantly decreases when cells are treated with CRISPR-Cas9 system combined with ADGN-PBN or with polyplus JetMessenger transfection agent compared to non-treated cells. CRISPR-Cas9 delivered by ADGN-PBN showed a gene editing efficiency of 40%. The resulting q-PCR signal for each condition was depicted relative to the maximum q-PCR signal obtained in the wild type condition. This result confirms the results obtained previously using the T7E1 mismatch detection assay and suggests that the CRISPR-Cas9 RNA delivered by ADGN-PBN induced gene editing at the targeted Luc sites in A549-Luc cell line.

CRISPR-Cas9 delivered by ADGN-PBN induced mainly one nucleotide insertion

In order to analyze the nature and the frequency of mutations induced by CRISPR-Cas9, genomic regions spanning the target Luc sites of isolated clones were PCR amplified and subjected to conventional capillary (Sanger) sequencing (Figure 52). Direct sequencing of PCR amplicons containing biallelic, heterozygous, or chimeric mutations resulted in superimposed sequencing chromatograms, which made it difficult to determine specific mutation types and mutation frequency in the clones generated after ADGN-PBN containing CRISPR-Cas9 system. To resolve this problem, tracking of indels by decomposition (TIDE) method was used to rapidly determine the mutated allelic sequences using the sequencing file (.ab1 format) with superimposed chromatograms [START_REF] Brinkman | Easy quantitative assessment of genome editing by sequence trace decomposition[END_REF], thus avoiding tedious and expensive cloning and multi-clone sequencing for mutation analysis.

TIDE analysis requires different information: a control sequence data file (obtained from nontreated A549-luc cells), a sample sequence data sample file (obtained from the CRISPR-Cas9 edited cells after ADGN-PBN transfection) and a sgRNA sequence. TIDE software aligns the non-treated control sequence to the sgRNA to determine the expected Cas9 cutting site and the experimental sample sequence is aligned to the control sequence in order to determine any offset between the two sequence reads. Alignments are done using the standard Smith-Waterman method. The software then uses the peak heights for each base, as determined by the sequence analysis software provided by the sequencing company (Genewiz) to determine the relative abundance of nucleotides over the length of the whole sequence trace. TIDE results revealed that DNA samples from CRISPR-Cas9 edited cells showed a sequence trace after the break site consisting of a mixture of signals (several peaks for the same position) derived from insertions and deletions resulting after NHEJ repair (Figure 53A). 95% of these ADGN-PBN treated samples showed alteration of the target site while the remaining 5% were identical to the wildtype (A549-luc non treated cells). From the edited samples, 30% showed indel frequencies higher than 15%. Most alterations were 1 base insertion, 4 bp after the PAM sequence (Figure 53B). Representative results of the proportion of aberrant base signals along the sequence traces are shown on Figure 54A, confirming that the break site is located at the expected position and affects most of the region after the mutation. The analysis of the spectrum of indels and their frequencies results in an estimation of the relative abundance of every possible indel within the chosen size range. For instance, clone 49 has a 43.7% frequency of +1 insertion, corresponding mostly to a guanine (G) insertion and have a total indel frequency of 55.5% (Figure 54B). These results confirmed that ADGN-PBN delivering CRISPR-Cas9 system provides an efficient tool to generate gene editing in cancer cells. ADGN peptides interact with bigger RNA molecules such as Cas9 mRNA. They form stable complexes with the CRISPR-Cas9 RNA components and are able to induce cellular uptake of the system to then release the RNA cargo in the cancer cells. This allows the formation of the CRISPR-Cas9 machinery, preserving its functionality. CRISPR-Cas9 delivered by ADGN-PBN induces the formation DNA double strand breaks, which repair produce DNA indels that were observed as mismatched DNA heteroduplexes by the T7E1 method. Gene editing capacity was confirmed with a 40 % decrease of qPCR amplification of the Luc gene. The most frequent mutation induced by the system is the insertion of one guanine, four nucleotides after the Cas9 cutting site. After validation of ADGN-PBN capacity to deliver a functional CRISPR-Cas9 system, able to induce the knock out of an exogenous gene, we proceed to target the more challenging KRAS gene.

A B

ADGN-PBN mediates efficient CRISPR-Cas9 delivery in vivo

Afterwards, we sought to assess whether the effect of CRISPR-Cas9, delivered by the ADGN-PBN carrier, on luciferase knockout observed in vitro could be translated to in vivo models. A549-luc cells were orthotopically engrafted into the lungs of nude mice. Intravenous administration of CRISPR-Cas9 complexed within ADGN-PBN, with a peptide to RNA molar ratio of 20, was performed 16 days post-tumor implantation (Figure 55).

To investigate the impact of peptide quantity on CRISPR-Cas9 delivery efficiency in vivo, an additional group of mice received a tenfold higher peptide to RNA molar ratio of 200.

In both conditions, a consistent total of 10 µg of RNA was injected, with corresponding peptide quantities of 10 µg and 90 µg for the 20 and 200 molar ratios, respectively. Lung tumor bioluminescence level was monitored using in vivo bioluminescence imaging until 3 days post-administration of the CRISPR-Cas9 ADGN-PBN complex. Luciferase-expressing A549 cells were orthotopically implanted into the lungs of nude mice. Following tumor engraftment, the mice were allowed to harbor the tumors for a period of 16 days to allow for substantial growth. Subsequently, ADGN-PBN carrying the CRISPR-Cas9 system were administered intravenously. The mice were then monitored for an additional 72 hours to assess the in vivo bioluminescence levels as a measure of response to the treatment and gene editing activity.

Three experimental conditions were tested: the first group (n=5) received ADGN-PBN complexed with RNA at a peptide to RNA molar ratio of 20, the second group (n=5) received a higher peptide to RNA molar ratio of 200, and the control group (n=3) received no peptide injection. Luminescence signals were predominantly localized to the left and right lung regions after tumor implantation at various time points (Figure 56). In group 1, consisting of mice injected with ADGN-PBN complexed with CRISPR-Cas9 at a peptide to RNA molar ratio of 20, a nuanced evaluation of treatment outcomes revealed that two out of five mice exhibited a noteworthy reduction, surpassing 50%, in luminescence intensity at the orthotopic lung tumor site when compared to the luminescence signals observed prior to treatment initiation (Figure 56). This observed decline in luminescence intensity suggests a discernible impact of the CRISPR-Cas9 system delivered by ADGN-PBN in mitigating luciferase expression within the tumor microenvironment.

Interestingly, within group 2, a subpopulation of mice receiving ADGN-PBN at a higher peptide to RNA molar ratio of 200 demonstrated a superior efficacy in luciferase knockout. The dose-dependent enhancement in the efficiency of CRISPR-Cas9-mediated gene editing supports the hypothesis that the quantity of ADGN peptide, as reflected by the varying peptide to RNA molar ratios, plays a critical role in influencing the therapeutic outcomes in vivo. This dose-responsive behavior provides valuable insights into optimizing the delivery parameters for enhanced precision in gene editing within the in vivo context.

Importantly, the control group, which did not receive ADGN-PBN or CRISPR-Cas9 treatment, exhibited no significant decrease in luminescence activity, reinforcing the specificity and selectivity of the ADGN-PBN delivery system in targeting luciferase expression.

In summary, our comprehensive analysis of these in vivo experiments offers compelling evidence of the potential of ADGN-PBN as a robust and adaptable vector for the systemic application of the CRISPR-Cas9 RNA system. Furthermore, the observed dose-dependent response emphasizes the importance of optimizing the delivery parameters for achieving maximum therapeutic efficacy in future applications of this innovative gene-editing approach.

Results part 3: ADGN-PBN targeting the predominant KRAS mutations

Mutations on the KRAS oncogene are the main drivers of common cancers including pancreatic, colorectal and lung cancer. KRAS mutations remain unmarketable in human cancer, causing severe health burden and mortality worldwide. The aim of the last part of my project was to evaluate the therapeutic potential of ADGN-PBN delivering CRISPR-Cas9 for the specific target of KRAS predominant variants. We already validated the capacity of ADGN-PBN to complex with the CRISPR-Cas9 RNA components while preserving the functionality of the system. Now we evaluated its potential therapeutic effect by knocking out the G12V and G12D KRAS mutations (Figure 57). First, we used the qPCR-based mutation detection method as it is a reliable, unexpensive and highly sensitive technique to evaluate CRISPR-Cas9 knock out efficiency at the DNA level. Then, we evaluated the level of expression of mutated KRAS protein after CRISPR-Cas9 editing as well as its effect on key transduction proteins. Finally, we assessed the impact of targeting G12D and G12V KRAS with CRISPR-Cas9 delivered by ADGN-PBN on cell proliferation.

ADGN-PBN mediate KRAS mutant knock out

To target mutant KRAS alleles in cancer cells with the CRISPR-Cas9 system, we first screened for guide RNAs that specifically target the predominant KRAS mutations, but not wild-type KRAS. Pancreatic cancer cells (Panc-1 cells) expressing the G12D KRAS mutation (c.35G>A) were transfected with ADGN-PBN complexed with 0.5 µg Cas9 mRNA and 0.5 µg sgRNA targeting G12D KRAS. The efficiency of the system was evaluated using three different sgRNA: 12D1, 12D2 or 12D3. Similarly, we transfected colorectal cancer cells (SW480) expressing the G12V KRAS mutation (c.35G>T) with ADGN-PBN this time targeting G12V KRAS (12V1, 12V2 or 12V3 sgRNA). The genomic DNA of each cell population was extracted 48 hours after transfection. It was qPCR amplified using a set of primers that were specifically designed to target the KRAS mutant sequences. In parallel, a non-productive phosphate-modified oligonucleotide complementary to the WT KRAS sequence was used to minimize possible amplification of mismatched products derived from the wild-type allele; ensuring high specificity of the system. The amplification of an internal control sequence was performed in a different reaction for each assay (Figure 58) to normalize the qPCR reaction efficiency.

Figure 58. Schematics of the methodology employed for the qPCR-based mutation detection method used for the relative quantification of G12D and G12V KRAS mutation after treatment with ADGN-PBN complexed with CRISPR-Cas9 [START_REF] Thierry | Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA[END_REF].

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Results showed that the efficiency of the system to generate indels on the targeted G12D KRAS mutant in Panc-1 cells was dependent on the sgRNA used (Figure 59A). A maximum KRAS expression decrease efficiency of 28% was reached after transfection with ADGN-PBN complexed with Cas9 mRNA and sgRNA 12D3 compared to non-treated cells. A decrease of 21% was also observed after transfection with ADGN-PBN containing sgRNA 12D2 compared to non-treated cells. No significant difference was obtained after transfection with sgRNA 12D1 compared to non-treated cells. It has to be noticed that a 20% KRAS expression decrease was observed if the results are compared to the ADGN-PBN containing the non-target sgRNA control.

In contrast, the efficiency of the system to target the G12V KRAS mutation on SW480 cells was close to 65% independently of the sgRNA used (Figure 59B). As expected, ADGN-PBN complexed with a non-target sgRNA control and Cas9 mRNA did not affect the qPCR amplification for none of the mutations. The qPCR amplification of the specific mutated KRAS fragments showed a shift in the qPCR amplification curve for both mutations. Figure 59C and D show representative amplification curves of the target KRAS gene after treatment with ADGN-PBN complexed with CRISPR-Cas9 and sgRNA 12D3 and 12V3 respectively. The relative quantification of the qPCR amplification was done using the relative standard curve method. This approach gives highly accurate quantitative results because unknown sample values are interpolated from the standard curves. The genomic DNA of from Panc-1 cells containing the G12D KRAS mutation and from SW480 cells containing the G12V KRAS mutation was serially diluted to determine the regression curves for the specific G12D or G12V primer sets as well as for the internal control primer sets (See Figure 72 on annexes section).

ADGN-PBN alter the expression of key signal transduction proteins and mutated KRAS

After validating the efficiency of all the tested sgRNA that specifically target G12V or G12D KRAS mutations, the level of expression of the specific mutated KRAS proteins and the effect on key signal transduction proteins were analyzed by western blot. The level of GAPDH was used as a loading control. The results showed a decreased of G12D KRAS expression level in CRISPR-Cas9 edited Panc-1 cells (Figure 60A) for all the sgRNA tested. Quantification analysis showed that lower G12D KRAS expression levels were obtained when sgRNA 12D3 was used, reaching a 50% decrease compared to non-treated cells (Figure 60C). The level of KRAS editing efficiency observed with the qPCR based mutation detection method (Figure 59A) correlates with the expression level of mutant G12D KRAS in the same conditions. Similarly, a 40% decrease of mutant G12V KRAS protein expression levels in SW480 cells is observed after treatment with ADGN-PBN complexed with CRISPR-Cas9 targeting G12V KRAS mutation (Figure 60B). No significant difference was observed within the edited groups using different sgRNA sequences (Figure 60D). Eukaryotic cells use a limited number of signal transduction pathways to integrate information from extracellular stimuli and to regulate cellular decisions such as proliferation, differentiation, and apoptosis. In particular, the PI3K/AKT and Ras/MEK/ERK pathways are implicated in the control of cell growth and proliferation in many different cell types (Figure 27). The effect on downstream key signal transduction pathways after CRISPR-Cas9 editing was analyzed by looking at the phosphorylation levels of ERK and AKT proteins on both Panc-1 and SW480 cells (Figure 61). This analysis was performed using the ADGN-PBN complexes that show the best mutated KRAS downregulation in the previous experiment.

After transfection of Panc-1 cells by ADGN-PBN complexed with Cas9 mRNA and 12D3 sgRNA, the levels of expression of AKT and ERK proteins remained constant. In contrast, phosphorylation levels of AKT and ERK proteins decreased in 44% and 47% respectively, compared to non-treated cells (Figure 62). Cells transfected with non-target sgRNA control showed same phosphorylation levels of AKT and ERK as non-treated cells. Panc-1 cells transfected with CRISPR-Cas9 RNA components delivered by polyplus JetMessenger showed no difference in expression of ERK or AKT, nor the phosphorylated forms of the proteins, suggesting no gene editing of the system delivered by this transfection vector.

Similarly, SW480 cells transfected with ADGN-PBN delivering CRISPR-Cas9 targeting G12V KRAS mutant had a decrease of 25% and 45% on the phosphorylation levels of AKT and ERK compared to non-treated cells or cells transfected with the control particles ADGN-PBN complexed with a non-target sgRNA (Figure 63). Polyplus JetMessenger transfection reagent showed higher efficiencies for this cell line, decreasing the phosphorylation levels of AKT and ERK by 45% and 50% respectively. These results demonstrated that in both cell lines, the treatment with ADGN-PBN reduced the expression levels of the targeted KRAS mutants and the downstream pERK and pAKT which are key proteins for the signaling pathways promoting cell growth, proliferation, and survival that finally leads to cancer development.

ADGN-PBN treatment impact cell proliferation

Next, we evaluated the effect of ADGN-PBN complexed with CRISPR-Cas9 targeting the G12D and G12V KRAS mutations on cell proliferation. The CellTiter Glow system was used to monitor Panc-1 and SW480 cell proliferation over a period of five days after transfection with ADGN-PBN complexed with CRISPR-Cas9 targeting G12D and G12V respectively. The ADGN-PBN complexed with sgRNA showing the best gene editing efficiencies in previous tests were evaluated for each mutation (sgRNA 12D2, 12D3, 12V2 and 12V3).

Results showed that the confluence of non-treated Panc-1 cells increased 4.6 times over a 5 days period while CRISPR-Cas9 edited Panc-1 cells showed an increase of only 2.4 and 2.2 times for sgRNA 12D1 and 12D2 respectively (Figure 64A). Similarly, non-treated SW480 cells increased cell proliferation by 4.8 times while CRISPR-Cas9 edited SW480 cells increased only 3.3 and 3.2 times for 12V2 and 12V3 sgRNA. These results confirmed that ADGN-PBN complexed with CRISPR-Cas9 targeting G12D and G12V predominant KRAS mutation can indeed decreased cell proliferation rate compared to non-treated cells. These results are in correlation with the decrease in AKT and ERK phosphorylation levels observed previously and that impacts cell proliferation. To date, there is no efficient therapeutic strategy to target the most common gene mutation associated to human cancer, KRAS. For the first time, a non-viral vector is used to deliver CRSIPR-Cas9 for the direct targeting of G12D and G12V predominant KRAS mutations. The system showed 50% and 40% of gene editing efficiency for each mutation respectively. Moreover, the knock-out of mutated KRAS, via CRISPR-Cas9, exerted a regulatory effect on downstream proteins that finally leaded to a decrease in cell proliferation. ADGN-PBN complexed with CRISPR-Cas9 RNA components showed promising potential as a new therapeutic strategy for KRAS associated cancers treatment.

ADGN-PBN mediate in vivo gene editing of G12D and G12V KRAS mutations

In order to comprehensively evaluate the therapeutic potential of ADGN-PBN in selectively targeting the frequently encountered G12D and G12V mutations within the KRAS oncogene in an in vivo setting, a series of preliminary experiments were meticulously conducted by Divincell. Female Balb/C nude mice, aged 6 weeks, were subjected to subcutaneous xenografts of human pancreatic cancer cells (Panc-1) and human colorectal cancer cells (SW403). Upon achieving a tumor size of 100 mm 3 , the mice were randomly divided into four distinct groups and subjected to treatment with ADGN-PBN, encapsulating the RNA components of CRISPR-Cas9 with a specific targeting of either the G12D or G12V KRAS mutation. The therapeutic regimen included intravenous administrations on days 1 and 7, with a peptide to RNA molar ratio of 200 and at doses of both 0.5 mg/kg or 1 mg/kg. Rigorous monitoring of tumor dimensions occurred on day 0 and subsequently at 5-day intervals (Figure 65). To establish a baseline, saline solution and ADGN-PBN containing a sgRNA targeting the G12C mutation were implemented as control groups. The findings underscored the robust efficacy of ADGN-PBN in effecting targeted editing of the G12D KRAS mutation in mice xenografted with Panc-1 cells, resulting in a notable prevention of tumor growth across both the 0.5 mg/kg and 1 mg/kg doses (Figure 66A). The treatment exhibited well tolerability, as evidenced by the absence of any appreciable weight loss in the treated mice (Figure 66B).

Parallelly, ADGN-PBN directed towards the G12V mutation effectively reduced tumor growth at both dosage levels (Figure 67A), maintaining a favorable safety profile with no observed weight loss (Figure 67B). Impressively, ADGN-PBN showcased efficient suppression of tumor growth when addressing the prevalent KRAS mutations on codon 12, achieving pronounced outcomes following merely two intravenous injections. Noteworthy is the evident dose-response relationship exhibited by both ADGN-PBN formulations targeting G12D and G12V mutations, further reinforcing their robust efficacy in an in vivo context. Importantly, all treatment regimens demonstrated exceptional tolerability, with no discernible adverse effects recorded.

In opposition, the control ADGN-PBN formulation targeting the G12C KRAS mutation exerted no discernible impact on tumor growth, firmly establishing the specificity and precision of the ADGN-PBN-mediated CRISPR-Cas9 delivery system. Collectively, these comprehensive in vivo findings offer compelling evidence of the therapeutic promise of ADGN-PBN in precisely targeting and modulating the KRAS mutations associated with codon 12, thereby advancing our understanding of the potential clinical applications of this innovative CRISPR-Cas9 delivery platform.

Discussion and perspectives

CRISPR-Cas9 system is a revolutionary gene-editing technology that has gained significant attention and excitement in the scientific community. However, despite its immense potential, CRISPR-Cas9 faces several notable challenges that have limited its widespread clinical use. One major challenge lies in effectively delivering the CRISPR-Cas9 components into cells and tissues, particularly in vivo. Delivering CRISPR-Cas9 components in their RNA forms presents an optimal approach, striking a delicate balance between expeditious expression and effective gene editing, all while ensuring costeffectiveness, high efficiencies, and eliminating the risk of DNA integration into the host genome. However, RNA components are more sensitive to nuclease degradation on bloodstream and require good protection from the delivery vehicle.

One of the primary obstacles encountered by the system involves the toxicity associated with the delivery of Cas9-nuclease and guiding RNAs into targeted cells. Toxicity arises during the introduction of CRISPR-induced double-stranded breaks, which can activate programmed cell death known as apoptosis. The occurrence of CRISPR-induced apoptosis raises safety concerns that need careful attention when contemplating the use of these gene editing systems [START_REF] Uddin | CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future[END_REF].

Enache et al. uncovered another significant obstacle associated with CRISPR/Cas9 systems. Their research revealed that the expression of Cas9 in human cell lines could result in the appearance or amplification of inactivating p53 mutations. They specifically demonstrated that p53 was among the top 4% of genes prone to mutation following the introduction of Cas9. Moreover, they found that Cas9 expression had a specific preference for inducing mutations that deactivate the p53 protein. As a result, the authors strongly recommend assessing the p53 status of cells following the introduction of Cas9, particularly in p53-wild type cells [START_REF] Enache | Cas9 activates the p53 pathway and selects for p53inactivating mutations[END_REF]. Therefore, it is of utmost importance to verify the presence of a functional p53 both prior to and following the implementation of CRISPR-Cas-based genome editing techniques.

Furthermore, the potential off-target effects of the CRISPR-Cas9 technique on the genome have raised significant concerns. Any unintended modifications to regions of the genome that were not intended to be altered can lead to undesired consequences or complications in the long term. While efforts can be made to minimize the risk of off-target editing through optimizing the concentration of the Cas9 enzyme and the duration of its expression, it does not completely eliminate the possibility of off-target editing. These offtarget effects manifest as unintended mutations, such as insertions, deletions, and point mutations, that have been reported in human cell studies. Additionally, there is a possibility of mismatching between the single guide RNA (sgRNA) and the target sequences due to their relatively short length. The larger the genome, the higher the risk of the sgRNA strands complementing off-target sites. Therefore, it is crucial to address these concerns and mitigate the potential off-target effects when utilizing the CRISPR-Cas9 system for genome editing [START_REF] Javed | CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms[END_REF][START_REF] Redman | What is CRISPR/Cas9? Archives of Disease in Childhood -Education and Practice[END_REF].

Despite these challenges, several encouraging CRISPR therapeutics are emerging for clinical application (Table 10). The majority of these clinical approaches involve the ex vivo transfection of the CRISPR-Cas9 system. However, Intellia Therapeutics stands out by utilizing a non-viral delivery approach using lipid nanoparticles for the systemic administration of CRISPR-Cas9. This innovative strategy is being employed for the treatment of TTA polyneuropathy and Hereditary angioedema.

Table 10. Clinical trials for disease gene editing with CRISPR-Cas9 [START_REF] Carroll | RNA in Therapeutics : CRISPR in the Clinic[END_REF]. CRISPR-Cas9 system is currently being investigated as a potential option for editing cancer mutations. Cancer is a highly complex disease that presents significant challenges in treatment, including acquired drug resistance and adverse side effects that greatly impact patients' quality of life. The search for personalized and sustainable therapy approaches for cancer has been ongoing for many years. Conventional treatments like chemotherapy and radiation therapy are still widely used, although they often cause damage to healthy organs and impair the immune system, leading to reduced life expectancy [START_REF] Pérez-Herrero | Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy[END_REF]. Their severe and sometimes lethal side effects have prompted the need for better therapeutic alternatives. Newer options like immunotherapy have shown promise but are often insufficient as standalone treatments, particularly in later stages of cancer where combination therapies with chemotherapy are often required [START_REF] Schirrmacher | From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment (Review)[END_REF].

The KRAS oncogene, which is mutated in a significant percentage of pancreatic, colorectal and lung cancers, plays a crucial role in the development of these aggressive forms of cancer, which have substantial public health implications and high mortality rates [START_REF] Abubaker | Prognostic significance of alterations in KRAS isoforms KRAS-4A/4B and KRAS mutations in colorectal carcinoma[END_REF][START_REF] Waters | KRAS: The Critical Driver and Therapeutic Target for Pancreatic Cancer[END_REF]. Consequently, KRAS has become an important target for therapeutic intervention in these cancer types.

The targetability of KRAS has presented significant challenges. The mutations in KRAS not only drive tumor progression but also play a crucial role in maintaining the tumor microenvironment, making it a complex therapeutic target. Despite persistent efforts, direct targeting of KRAS has been unsuccessful. This can be attributed to the resistance 122 of KRAS-mutated tumors to currently available clinical therapies. KRAS operates within a complex biochemical network, interacting with numerous upstream and downstream effectors, which has made it difficult to identify an effective strategy for inhibiting its activation. Approaches that aimed to target the effectors of KRAS have proven inadequate in effectively restraining the mutated KRAS and achieving desirable outcomes. Consequently, studies have shifted towards a combination therapy approach, which necessitates direct targeting of KRAS itself to achieve a comprehensive therapeutic response (P. [START_REF] Liu | Targeting the untargetable KRAS in cancer therapy[END_REF].

The incorporation of CRISPR platforms into cancer therapeutics holds promise as a novel approach. However, there has been limited research focused specifically on targeting KRAS using CRISPR systems. These studies have provided valuable insights into potential therapeutic strategies for targeting the main KRAS mutations, such as G12D and G12V, using CRISPR-Cas9. Nevertheless, further research is necessary to translate these findings into clinical applications. One key aspect highlighted by these studies is the importance of finding a suitable vector for the safe and efficient delivery for CRISPR-Cas9 in vivo.

Nanomaterials have emerged as a promising alternative to viral vectors for CRISPR-Cas9 delivery, offering distinct advantages. A wide range of nano-delivery systems have been developed specifically for CRISPR-Cas9, encompassing cationic liposomes, lipid nanoparticles, cationic polymers, gold nanoparticles, and peptide-based nanoparticles. These diverse nanomaterials have demonstrated their efficacy and versatility in facilitating the delivery of CRISPR-Cas9 components to target cells or tissues. By harnessing the unique properties of nanomaterials, such as their tunable surface charge, biocompatibility, and high loading capacity, researchers have been able to overcome some of the limitations associated with viral vectors. The utilization of nanomaterialbased delivery systems opens new possibilities for precise and efficient gene editing, advancing the field of CRISPR-based therapeutics.

During my PhD research, I focused on investigating the feasibility of using peptide-based nanoparticles for the transient delivery of CRISPR-Cas9 components in their RNA form to target exogenous or endogenous genes. We have developed novel ADGN cell-penetrating peptides that have been optimized to self-assemble with nucleic acids, forming stable peptide-based nanoparticles. This thesis shows that transfection of human tumoral cells using CRISPR-Cas9 complexed to ADGN-PBN in a non-covalent manner is possible and it is comparable in luciferase genome editing efficiency to transfection of Cas9/sgRNA with a commercial transfection kit called polyplus JetMessenger. This is the first study demonstrating that PBN are also able to efficiently carry functional CRISPR-Cas9 system for gene editing of KRAS.

We specifically selected peptide-based nanoparticles due to their potential to overcome various limitations associated with CRISPR-Cas9 delivery. PBN possess several advantageous characteristics, including their ability to encapsulate large mRNA encoding for the Cas9 protein and single guide RNA, and to efficiently traverse cellular barriers. Additionally, PBN provide protection to the cargo from degradation pathways such as 123 proteases, RNAses, lysosomes, and macrophages. Furthermore, they ensure the integrity of the guide RNA and functional Cas9 proteins, enabling effective genome editing. Importantly, compared to other nanocarriers, we demonstrated that they exhibit low toxicity. It has been shown in previous work performed by Divincell company, that they also induce minimal immunogenic responses, and can be easily broken down into amino acids, making them a promising platform for safe and effective CRISPR-based therapies.

These ADGN peptides possess an interesting secondary amphipathic conformation, which allows for initial electrostatic interactions between the positive charges of the peptides and the negative charges of the nucleic acids. Subsequently, the complex is stabilized through hydrophobic interactions. This unique secondary amphipathic structure enables ADGN peptides to efficiently encapsulate the nucleic acids, resulting in the formation of stable noncovalent nanoparticles. While the use of cell-penetrating peptides as potential delivery systems for nucleic acids through non-covalent strategies has been extensively explored, this study represents the first reported use of PBNs for delivering the CRISPR-Cas9 system in its RNA form.

During the optimization of PBN formulation, I observed that the positive charges of the peptides play a crucial role in complexing with nucleic acids and influence the final surface charge of the PBN. By adjusting the peptide to RNA ratio, we have developed PBNs with a final zeta potential ranging from -12mV to +10mV. Although ongoing debates exist in the field of nanomedicine regarding the surface charge of therapeutic nanoparticles and its impact on opsonization in the bloodstream, it is worth noting that most PBNs have been predominantly characterized as positively charged [START_REF] Boisguérin | Peptide-based nanoparticles for therapeutic nucleic acid delivery[END_REF]. These positively charged PBNs have demonstrated effective delivery of nucleic acids even in the presence of serum, suggesting that the positive surface charge does not pose significant limitations in vitro. However, to gain a comprehensive understanding, further exploration and evaluation of the in vivo behavior and potential implications of surface charge on the fate and performance of therapeutic nanoparticles are important areas of research.

ADGN-PBN were optimized to achieve a size distribution of approximately 100 nm. This particular size range is considered optimal for therapeutic applications, as it falls above the 6 nm threshold that would lead to filtration by the kidneys, while staying below the 200 nm limit associated with rapid uptake by the liver and spleen due to complement activation. By maintaining a size within this range, nanoparticles demonstrate improved circulation time and biodistribution properties (Z. [START_REF] Zhao | Effect of physicochemical and surface properties on in vivo fate of drug nanocarriers[END_REF]. The homogeneity of ADGN-PBNs was carefully assessed, and reproducible measurements of the polydispersity index consistently remained below the threshold of 0.3. This level of homogeneity is considered acceptable for therapeutic applications, ensuring consistent and reliable performance [START_REF] Danaei | Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems[END_REF]. It is important to highlight the critical role of standardizing the complexation protocol and optimizing the physicochemical properties of the PBNs during the project. Various parameters, such as agitation speed and the rate of peptide addition to the solution, were carefully controlled, as they significantly influenced the properties of the nanoparticles.

Furthermore, our observations indicated that the compact complexation between ADGN peptides and the mRNA provided effective protection against degradation by RNAse enzymes. This finding aligns with previous reports demonstrating the ability of PBNs to safeguard nucleic acid cargo [START_REF] Jang | CRISPR prime editing for unconstrained correction of oncogenic <em>KRAS</em> variants[END_REF]. The CRISPR-Cas9 RNA cargo remained protected within the ADGN-PBNs even in the presence of up to 100 ng/ml of RNAse A, which is the reported concentration of nucleases in the bloodstream [START_REF] Herriott | Nucleases and Their Inhibitors of Human in the Cellular Blood ' Components Separation of the Cellular Elements of Blood[END_REF].

After confirming the ability of ADGN-PBN to encapsulate large mRNA molecules and deliver them to various cancer cells, the next step involved the encapsulation of the CRISPR-Cas9 system. This presented a particular challenge in optimizing the properties of ADGN-PBN after incorporating the additional RNA molecule (sgRNA), which is significantly smaller in size and tends to increase the polydispersity of the system.

Once the physicochemical properties were optimized, it was essential to assess the interaction of ADGN-PBN containing the CRISPR-Cas9 components with cells. It was observed that ADGN-PBN accumulated in the cell membrane and then underwent penetration, eventually releasing the RNA cargo into the cytosol. This demonstrated their capability to co-deliver Cas9 mRNA and sgRNA. While PBN utilizing CADY peptide have been reported to be internalized through direct translocation [START_REF] Vertical Rydström | Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles[END_REF], our ADGN-PBN primarily underwent internalization via endocytosis.

As a first proof of concept, I investigated the ability of ADGN-PBN to deliver intact RNA cargo into cells, able to form a functional CRISPR-Cas9 machinery for gene editing. I targeted the exogenous luciferase reporter gene in human cancer cells for this purpose. Optimization of the Cas9 to sgRNA ratio was found to be crucial for achieving functionality of the system. It was observed that sgRNA acted as the limiting reagent, and a 1:1 mass ratio of Cas9 mRNA to sgRNA was required for optimal results. Although the optimal ratio may vary depending on the transfection conditions and target cells, a 1:1 mass ratio has been suggested for both mRNA and RNP delivery modes [START_REF] Kouranova | CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos[END_REF][START_REF] Qiu | Lipid nanoparticlemediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3[END_REF]. Total quantity of Cas9 mRNA was an important parameter as ADGN-PBN loaded with more than 2 μg of Cas9 mRNA induced cytotoxicity due to the high concentration of Cas9 protein, as previously reported [START_REF] Uddin | CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future[END_REF].

ADGN-PBN efficiently allowed the knockout of the exogenous luciferase gene using CRISPR-Cas9. The editing process demonstrated a dose-dependent reduction in luciferase expression, with a maximum 60% decrease observed when cells were transfected with ADGN-PBN containing 0.5 μg of Cas9 mRNA and 0.5 μg of sgRNA at a 1:1 peptide to RNA weight ratio. Previous studies have reported similar dose-dependent luciferase knockouts in Hela cells, which stably expressed both Cas9 mRNA and luciferase, when transfected with zwitterionic amino lipid nanoparticles encapsulating sgRNA targeting luciferase. In those studies, optimized nanoparticles achieved up to a 90% decrease in luciferase expression (J. B. Miller et al., 2017).

While our particles exhibited slightly lower efficiency, it is important to consider that we evaluated the total cell population (transfected and no transfected cells), therefore the evaluation of knock out efficiency is underestimated compared to other studies where transfected cells are selected before gene efficiency studies. In addition, it is possible that only one allele of luciferase gene could be targeted and not the second allele, allowing a lower luciferase activity compared to non-treated cells. Moreover, we co-delivered both Cas9 mRNA and sgRNA, while they deliver sgRNA in cells stably expressing Cas9 protein.

Another study using lipid nanoparticles to deliver CRISPR RNA components for base editing showed efficiencies of 60% when targeting the PCSK9 gene after selection of transfected cells. Importantly, we did not observe a decrease in luciferase expression levels when Cas9 mRNA was administered with a non-targeting sgRNA control, highlighting the activity of Cas9 only when guided by the RNA sequence to the target DNA. ADGN-PBN reached the same gene editing efficiency as the commercially available polyplus JetMessenger transfection reagent in A549 cells and reached 60% efficiency on MCF7 cells that did not show any significant decrease in luciferase activity after transfection with polyplus JetMessenger following the manufactures instructions.

To validate the CRISPR-Cas9-induced knockout of luciferase, we employed two methods: the gold standard T7E1 assay, commonly used for detecting CRISPR-induced indels in the target DNA, and an innovative qPCR-based mutation detection method. Both methods confirmed the impact of CRISPR-Cas9 delivered by ADGN-PBN on the specific target DNA.

The introduction of indels in the DNA sequence due to Cas9 activity generated heteroduplexes, resulting in a 60% cleavage efficiency of the T7E1 enzyme. Similarly, editing of the luciferase gene led to changes in the DNA sequence, which reduced primer affinity and subsequently resulted in decreased qPCR amplification. ADGN-PBN showed high gene editing efficiencies compared to previously reported PBN delivering the CRISPR-Cas9 RNP complex, were gene editing efficiencies between 3% and 40% were observed using the T7E1 mismatch detection assay [START_REF] Liu | Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition[END_REF][START_REF] Ramakrishna | Gene disruption by cellpenetrating peptide-mediated delivery of Cas9 protein and guide RNA[END_REF].

Sequencing analysis plays a crucial role in assessing the outcomes of gene editing performed by the CRISPR-Cas9 system, allowing for the determination of the induced mutation type. It is important to note that mutagenic repair processes can result in substantial deletions, chromosomal translocations, and the integration of delivery reagents at both on-target and off-target sites. Consequently, these outcomes pose significant safety concerns for potential clinical applications. In light of this, once the activity of the CRISPR-Cas9 system on the luciferase target gene was confirmed, we conducted sequencing analysis to investigate the specific effects of the system on the DNA. The results revealed that CRISPR-Cas9 induced indels in 95% of the sequenced clones. Among the observed DNA modifications, the most frequent was the insertion of a single guanine nucleotide four positions upstream of the PAM sequence. It is important to note that for a more robust statistical analysis, a larger sample of clones would be required.

The mutational outcomes of CRISPR-Cas9 can vary depending on the cell line used. Cells differ in their repair processes and DNA sequences, which both influence the outcomes of double-strand break repair (Gallagher & Haber, 2018). However, previous studies have shown a significant preference of CRISPR-Cas9 to generate single-nucleotide insertions in yeast and humans (Lemos et al., 2018). This observation could be explained by a model in which the Cas9 protein remains bound to the PAM-proximal side of the cut, while DNA polymerase fills the staggered one-nucleotide overhang on the PAM-distal side, followed by re-ligation via the non-homologous end joining pathway (Shou et al., 2018).

Furthermore, I assessed the translational potential of CRISPR-Cas9 delivered by the ADGN-PBN, from in vitro to in vivo models using orthotopically engrafted A549-luc cells in the lungs of nude mice. The evaluation of treatment outcomes revealed a reduction in luminescence intensity at the orthotopic lung tumor site in mice injected with ADGN-PBN complexed with CRISPR-Cas9 at a 20:1 peptide to RNA molar ratio. Interestingly, a subpopulation of mice treated with a higher peptide to RNA molar ratio of 200:1 demonstrated superior efficacy in luciferase knockout. The control group exhibited no significant decrease in luminescence activity, underscoring the specificity and selectivity of the ADGN-PBN delivery system.

The comprehensive in vivo analysis provided evidence for the potential of ADGN-PBN as a versatile vector for the systemic application of CRISPR-Cas9 RNA, with the observed dose-dependent response highlighting the crucial role of optimizing delivery parameters for achieving maximum therapeutic efficacy. These findings offer valuable insights for the future refinement and application of this innovative gene-editing approach in clinical settings.

During the final phase of my PhD project, I dedicated my efforts to assessing the capability of ADGN-PBN, encapsulating CRISPR-Cas9, to target the predominant G12D and G12V mutations in the KRAS gene. Through qPCR mutation detection, I confirmed the system's ability to successfully edit this challenging endogenous gene. The editing efficiency was determined to be 28% for the heterozygous G12D KRAS mutant-expressing Panc-1 cells and 65% for the homozygous G12V KRAS mutant-expressing SW480 cells. The discrepancy in efficiency could be attributed to the varying transfection efficiency of ADGN-PBN in each cell line or cell-specific factors such as the levels of endogenous repair mechanisms, DNA accessibility, and gene expression. Previous studies have demonstrated that CRISPR-Cas9 targeting of the G12D and G12V mutations in KRAS resulted in higher gene editing efficiencies following puromycin selection of transduced cells. Notably, SW480 cells exhibited an indel frequency of 80%, while LS513 cells, expressing the G12D KRAS mutant, displayed a 36% indel frequency (W. Kim et al., 2018). Heterozygosity can affect the editing efficiency by influencing the competition between the wild-type and edited alleles during the repair process. The repair mechanisms, such as non-homologous end joining or homology-directed repair, may favor one allele over the other, leading to variations in the editing efficiency between the alleles. Kim et al. reported lower gene editing efficiency of KRAS mutant in heterozygous SW403 cells (30%) compared to the homozygous SW480 cells (80%) expressing the same G12V KRAS mutations (W. Kim et al., 2018).

Furthermore, the targeting of the G12D KRAS mutant gene in Panc-1 cells with ADGN-PBN resulted in a significant impact on cell proliferation, reducing it by 50%. Similarly, SW480 cells harboring the G12V KRAS mutation exhibited a 30% decrease in cell proliferation after ADGN-PBN treatment. These findings align with a study by Lee et al., where they observed a 30% decrease in cell proliferation in SW480 cells following CRISPR-Cas9 gene editing of the G12V KRAS mutation. However, they reported no significant effect on cell proliferation in SNU407 and AsPC-1 cells expressing the G12D KRAS mutation after CRISPR-Cas9 treatment.

My results revealed that the DNA editing of the mutated KRAS gene led to altered expression levels of the mutant protein. Panc-1 cells exhibited a 50% reduction in G12D mutant KRAS expression, while SW480 cells showed a 40% decrease in G12V mutant KRAS expression. Notably, the knockdown of mutated KRAS also impacted the phosphorylation levels of AKT and ERK, crucial downstream signaling proteins. Previous studies have demonstrated that MRTX-1133, a noncovalent inhibitor specifically designed to target KRAS G12D, can effectively deactivate the mutated KRAS protein and inhibit ERK phosphorylation (X. [START_REF] Wang | Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12DInhibitor[END_REF]. However, its effect on AKT phosphorylation remains unreported. Likewise, targeted knockdown of KRAS G12D using the CRISPR-Cas13 system demonstrated a significant reduction in the expression of phosphorylated-ERK and phosphorylated-AKT proteins in pancreatic cancer cells.

Limited studies have directly targeted the G12V KRAS mutant, and their evaluation did not extend to the impact on downstream protein signaling pathways.

Preliminary experiments were conducted in order to assess the in vivo efficacy of ADGN-PBN encapsulating the CRISPR-Cas9 system to target mutant KRAS. Female nude mice were xenografted with Panc-1 cells expressing the G12D mutation. The mice were intravenously injected with ADGN-PBN on day 1 and day 7 of the study. ADGN-PBN containing a RNA cargo dose of 0.25 mg/kg, demonstrated a significant 50% reduction in tumor size by day 50 compared to both the control group and the mice treated with ADGN-PBN containing a non-targeting sgRNA control. Encouragingly, when the dose was doubled to 5 mg/kg of RNA cargo, the tumor size did not exhibit any increase compared to the measurements taken on day 0. These preliminary results are highly promising; however, further confirmation with a larger number of animals per group is necessary to facilitate robust statistical analysis and validate the observed outcomes.

When addressing KRAS mutations in pancreatic cancer, an additional obstacle arises due to the challenging access to the tumor. PDA is characterized by a dense, fibrous stroma that acts as a barrier, impeding the effective delivery of therapeutic agents to the tumor site. This desmoplastic stroma in pancreatic cancer exerts physiological effects on the tumor, directly influencing the efficacy of therapeutic agents. Unlike many tumors that rely on the formation of new blood vessels (neo-angiogenesis), ductal pancreatic tumors have significantly lower vascularization compared to normal tissues, resulting in poor blood perfusion. Consequently, the inadequate perfusion of pancreatic tumors limits the successful transport of therapeutic agents into the tumor parenchyma. ADGN-PBN holds the potential to overcome this barrier due to its small size that enables passive accumulation within tumors, but mostly due to its YIGSR targeting motif. Active targeting has been shown to surpass the impact of nanoparticle size alone, greatly enhancing tumor-specific uptake in orthotopic pancreatic cancer. However, precise studies should be performed to confirm the improved targeting of ADGN-PBN to cells overexpressing the laminin receptor. Several experiments could be performed for this, including comparing in vitro cellular internalization of ADGN-PBN to PBN lacking the targeting motif by flow cytometry or confocal imaging. A competitive binding assay where cells are incubated with ADGN-PBN after excess exposure to the targeting ligand or a comparison of therapeutic effect of ADGN-PBN vs control PBN in cells overexpressing the laminin receptor.

Future studies should be conducted to validate the precise effects of CRISPR-Cas9 gene editing on the targeted KRAS gene. It is crucial to confirm the gene editing capacity in various cell lines that express the prevalent KRAS mutations. By performing sequencing analyses following ADGN-PBN treatment, we can elucidate the specific impact of CRISPR-Cas9 editing on the target gene and the nature of the indels induced. Moreover, additional in vivo studies are necessary to investigate important aspects such as biodistribution after systemic administration, potential therapeutic effects, and immune responses. Furthermore, comprehensive toxicology studies are essential to evaluate the possible adverse effects of ADGN-PBN mediated CRISPR-Cas9 therapy on different organs and systems. To facilitate translation to clinical applications, meticulous control over the physicochemical properties of the nanoparticles is required, particularly following the scale-up process. In this thesis, we have not considered the off-target effects of our gene editing. Although we have minimized potential off-target effects by utilizing mRNA Cas9, which exhibits transient expression, and employing a sgRNA with a unique target site in the genome with no significant similarity to other genes, it is imperative to conduct extensive off-target analysis, including genome-wide sequencing, to identify any potential off-target effects before moving any gene editing techniques toward the clinic.

To enhance the precision and minimize potential off-target effects, various strategies have been explored, including the utilization of modified Cas9 proteins or exogenous template DNA. In the initial stages of this project, we employed mRNA encoding for HYPA Cas9, a hyper-accurate variant of Cas9. HYPA Cas9 has undergone modifications in the REC3 domain, which enhances the level of homology required between the sgRNA and the target DNA to generate a double-strand break (DSB) [START_REF] Ikeda | High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes[END_REF]. However, we encountered challenges in obtaining consistent and reproducible results with this approach. One potential explanation for the inconsistent outcomes could be attributed to the production of mRNA through in vitro transcription of a plasmid DNA, as the yields and quality of the mRNA varied. To address this issue, we resolved the reproducibility problems by obtaining a high-quality Cas9 mRNA from a reliable supplier (TriLink BioTechnologies). This ensured the consistency and reliability of the Cas9 mRNA used in our experiments, leading to improved and dependable results. Further investigations and optimizations may be necessary to fully harness the potential of modified Cas9 proteins, such as HYPA Cas9, for minimizing off-target effects and achieving precise gene editing outcomes.

Accurate modification of the genome can also be accomplished through the simultaneous delivery of external donor DNA, which acts as a blueprint for homology-directed repair at the double-strand breaks created by Cas9. However, the occurrence of deletions and/or insertions resulting from the error-prone joining of Cas9-induced DSBs poses a challenge to achieving precise and error-free modifications of the genome. Additionally, alternative approaches have been proposed to indirectly enhance the efficacy of accurate gene editing by suppressing mutagenic end joining [START_REF] Schimmel | Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways[END_REF]. However, efforts to inhibit the key factors involved in non-homologous end joining, such as DNA-PKcs and LigIV, to reduce the mutagenic repair of Cas9-induced breaks have yielded inconsistent outcomes in gene editing, with results varying depending on the specific cell type and culturing conditions [START_REF] Yeh | Advances in genome editing through control of DNA repair pathways[END_REF]. The inconsistent impact on HDR when NHEJ is inhibited might be attributed to the presence of an alternative end-joining pathway known as theta-mediated end joining (TMEJ).

The utilization of ADGN-PBN-mediated CRISPR-Cas9 targeting of KRAS mutations presents a compelling alternative to existing chemotherapies, which often impose numerous adverse effects on patients. However, it is crucial to recognize that KRAS mutations should not be addressed in isolation due to the interdependent nature of genes and their role in cancer progression. Consequently, to facilitate the potential translation of this KRAS-targeting ADGN-PBN strategy to clinical applications, it should be combined with other relevant molecules involved in the associated pathways. For example, it could be used in combination with upstream signaling inhibitors such as cetuximab that has shown promising outcomes in clinical trials. When combined with the direct G12C KRAS inhibitor adagrasib, it exhibited an overall response rate of 43% and a disease control rate of 100%. Similarly, it could be combined with afatinib which resulted in an overall response rate of 30.3% and a disease control rate of 75.8% when combined with the G12C inhibitor sotorasib.

Furthermore, exploring the combination of ADGN-PBN targeting KRAS with immune checkpoint inhibitors like TNO155, an oral inhibitor of SHP2, is another possible strategy. TNO155 has being actively investigated in both frontline and refractory settings for nonsmall cell lung cancer. This approach holds promise and warrants further exploration to assess its efficacy and potential synergy with ADGN-PBN therapy. By integrating complementary therapeutic agents and strategies, such as upstream signaling inhibitors and immune checkpoint inhibitors, with the ADGN-PBN-mediated CRISPR-Cas9 targeting of KRAS mutations, we might enhance the therapeutic potential and broaden the scope of treatment options for patients with KRAS-mutant cancers.

The cost of utilizing CRISPR-Cas9 gene editing therapy for cancer is an important aspect that requires attention. Particularly as ensuring equitable access to this groundbreaking therapy is a fundamental concern. The introduction of Zolgensma, a newly FDA-approved gene therapy drug for spinal muscular atrophy, with a price tag of $2.125 million, offers some insight into the potential cost of gene editing techniques. However, it is worth noting that gene editing has the potential to provide a curative treatment, which should be taken into consideration when assessing costs over a patient's lifetime. In fact, when comparing the expenses associated with long-term patient care, gene editing may prove to be a more economical option. Furthermore, as gene editing continues to be more widely implemented, the costs are expected to decrease significantly.

Overall, this research highlights the potential of ADGN-PBNs as an effective delivery system for CRISPR-Cas9 components, enabling precise gene editing and providing new insights into targeting endogenous genes. ADGN-PBN offers a significant alternative to current treatments, as it holds the potential to be highly safe with minimal adverse effects for patients, while still maintaining important therapeutic benefits being cost-effective. Nevertheless, despite the remarkable efficacy in delivering therapeutic payloads into cells, there are still challenges to be addressed in achieving precise control over the physical properties of PBN, especially during the scale-up process. The findings of my work contribute to the growing body of knowledge in the field of gene therapy and provide a foundation for further advancements in the development of safe and efficient CRISPR-based therapeutic approaches. With continued research and development, peptide-based nanoparticles hold great promise in revolutionizing the field of gene therapy and precision medicine.

Materials and methods

Peptides and RNAs

ADGN-ava [Ac-YIGSR-Ava-LWRALWRLLRSLWRLLWK-NH2] and ADGN-peg2 [Ac-PEG2-LWRALWRLLRSLWRLLWKR-NH2] peptides were obtained from GENEPEP. Peptides are provided in powder and are stored at -80°C. It is recommended to prepare stock solutions of 550 µM. For this, the peptides should be equilibrated at room temperature for 30 minutes to prevent peptide hydration. Then, the peptide powder is solubilized by adding DMSO to obtain a concentration of 20 µL/mg of peptide. Finally, a 550 µM concentration is reached adding nuclease free water dropwise to the DMSO solution. The solution is then vortexed gently for 2 minutes at low speed and subsequently sonicated for 10 minutes. Peptide 550 µM stock solution can be stored at -80°C for one month or at 4°C for one week. Only one freeze-thaw cycle is recommended. ADGN-PBN are prepared with a peptide working solution at 0.5 µg/µl, prepared with 60% v/v ADGN-ava and 40% v/v ADGN-peg2.

The polyadenylated and capped spCas9 mRNA expressing a version of Streptococcus pyogenes SF370 Cas9 protein (CRISPR associated protein 9) was obtained from Trilink BioTechnologies (catalog # L-7606). Cas9 mRNA encodes for the Cas9 protein with an N and C terminal nuclear localization signal (NLS) which increases the frequency of delivery to the nucleus, thus increasing the rate of DNA cleavage. The fLuc mRNA encoding for the luciferase protein, originally isolated from the firefly Phothinus pyralis, was obtained from Trilink BioTechnologies (catalog # L-7602). Both mRNA sequences are capped using the CleanCap technology, a posttranscriptional capping method that results in the naturally occurring Cap1 structure, they are polyadenylated and optimized for mammalian systems, mimicking a fully processed mature mRNA. The different single guide RNAs were obtained from IDT (Integrated DNA Technology). The sequences of the sgRNA used are shown in the table below (Table 11).

Target gene Name of sgRNA Sequence

Luciferase Luc 5'-ACAACTTTACCGACCGCGCC-3' KRAS G12D 12D1 5'-AGTTGGAGCTGATGGCGT-3' KRAS G12D 12D2 5'-CTTGTGGTAGTTGGAGCTGA-3' KRAS G12D 12D3 5'-GTAGTTGGAGCTGATGGCGT-3' KRAS G12V 12V1 5'-TAGTTGGAGCTGTTGGCGT-3' KRAS G12V 12V2 5'-CTTGTGGTAGTTGGAGCTGT -3' KRAS G12V 12V3 5'-GTAGTTGGAGCTGTTGGCGT-3' Non-target Ctrl 5'-AAAUGUGAGAUCAGAGUAAU-3'

Nanoparticles formulation

Complex formation constitutes a critical step for the successful encapsulation and transfection of RNA and therefore, should be followed carefully. Briefly, peptide stock solutions were diluted with nuclease-free water to a final working concentration of 0.5 µg/µl. Peptide working solutions were then sonicated for 10 minutes in a water bath sonicator with 30 s ON/OFF intervals, to prevent aggregation and finalize the solubilization of the peptides. The ADGN-ava and ADGN-peg2 peptides were mixed to a 60/40 v/v ratio respectively, to prepare ADGN peptide working solution. 3 µg mRNA and 3 µg sgRNA were added to 132 µl of nuclease-free water on a glass container under sterile conditions. 12 µl of the ADGN peptide working solution (0.5 µg/µl) was added dropwise under constant agitation (400 rpm) to reach a peptide:RNA weight ratio of 1:1. The solution was then incubated at room temperature for 30 min to allow the formation of the complexed PBNs. Finally, the complex was diluted in 50% v/v serum-free medium before transfection. Although the peptide-RNA complexes are stable for 2 weeks at 4°C we suggest using them freshly prepared for each experiment to guarantee high efficiency and reproducibility.

Size distribution and zeta potential

Proton correlation spectroscopy and electrophoretic mobility were used to measure the average size distribution and surface charges (zeta potential) of ADGN-PBN complexed with RNA at different weight ratios. Dynamic light scattering was measured using a Zetasizer Nano ZS system (Malvern Instruments, UK) with a fixed scattering angle of 90º at 25°C. For water, 1.33 and 0.89 mPa.s were taken as refractive index and viscosity values, respectively.

Transmission electron microscopy (TEM)

Transmission electron microscopy was used to analyse the size, shape and morphology of ADGN-PBN complexed at peptide:RNA weight ratio of 1:1. Complexes were prepared as described above with 0.5 μg Cas9 mRNA and 0.5 μg sgRNA. Nanoparticles suspension was drop-cast onto a TEM grid and allowed to air dry at room temperature and cover in gold with a Balzer SDC 030. The TEM grid was transferred to the TEM Hitachi S800 instrument, and the images of nanoparticles were acquired.

Cell culture conditions and transfections

Human Panc-1 cells, a cell line isolated from a pancreatic carcinoma of ductal cell origin, SW480 cell line established from a primary adenocarcinoma of the colon and Hela human epithelial adenocarcinoma cells were purchased from the American Type Culture Collection (ATCC® CRL-1469™, CCL-228™ and CCL-2 ™ respectively) and were cultured in DMEM medium with 10% heat-inactivated FBS in a humidified incubator at 37°C and 5% CO2. Human lung adenocarcinoma H441 cell line and H358 bronchioalveolar barcinoma non-small cell lung cancer were purchased from the American Type Culture Collection (ATCC® HTB-174 and CRL-5807 respectively) and were cultured in RPMI medium with 10% heat-inactivated FBS in a humidified incubator at 37°C and 5% CO2.

Human non-small cell lung cancer A549 cells (ATCC® CCL-185™) stably transfected with pGL3 firefly luciferase (A549-luc) and human breast cancer cells MCF7 stably expressing luc2 luciferase (ATCC® HTB-22™) were obtained from Optimal platform (IAB Grenoble, France) and were cultured in RPMI culture medium or DMEM culture medium respectively. For both cell lines, the culture medium was supplemented with 2mg/ml of G418 (geneticin antibiotic) and 10% heat-inactivated FBS. Cells were kept in a humidified incubator at 37°C and 5% CO2.

One day before transfection, cells were plated at 60-70% confluence in 96 well plates (cell number ranging from 10000 to 12500 cells depending on the cell line) and incubated for 24 hours. Cells were washed with 100 μl of basic medium with no serum, 50 μl of ADGN-PBN with peptide:RNA weight ratio of 1:1 containing 0.5 µg Cas9 mRNA (6.7 nM) and 0.5 µg sgRNA (309 nM) were added and incubated for 10 minutes at 37°C. Then 50 μl of basic medium (no serum supplementation) was added and cells were incubated at 37°C for 2 hours. Finally, 100 μl of complete medium with 10% FBS was added, and cells were incubated for 48 hours before analysis. As a positive control for transfection, polyplus JetMessenger reagent was used according to the manufacturer's instructions. In addition, a non-specific sgRNA sequence with no target on the human or mouse genome was used.

MTS analysis

Cell viability was measures using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS assay) from Promega, according to the manufacture's instructions. Briefly, cells were seeded in 96-well plates and transfected with ADGN-PBN. 48 hours after transfection, 20 μl of MTS solution was added to each well. The plate was then incubated at 37°C for 1 hour in a humidified, 5% CO2 atmosphere. The absorbance was measured at 490 nm using a CLARIOstar Plus microplate reader (BMG Labtech).

Electrophoretic mobility shift assay

PBN complexes were prepared with 0.5 μg of Cas9 mRNA and increasing peptide:RNA weight ratios (0:1, 0.25:1, 0.5:1, 1:1 and 2:1) and were loaded on 1% agarose gel electrophoresis stained with 1X gelGreen solution, to verify the encapsulation capacity.

The bands corresponding to the RNA were scanned using the filter gelGreen by ChemiDoc™ MP Imaging System (Bio-Rad).

The ability of PBN to withstand the direct digestion of RNAse A was also determined by gel electrophoresis. To do so, RNA samples were were incubated with RNAse A in prescence or absence of the nanoparticles. ADGN-PBN were prepared with 0.5 μg of mRNA, 0.5 µg sgRNA and a peptide:RNA weight ratio of 1:1. ADGN-PBN were digested with 0, 10, 50, 100 and 500 ng/ml of RNAse A at 37°C for 30 minutes. After digestion, RNAse A was degraded using 1X RNAsecure™ RNase Inactivation Reagent (Thermo fisher scientific) according to the manufacturer instructions and subsequently, the PBN were incubated with 10 µl proteinase K (0.12 µg/µl) for 60 minutes at 50°C in order to dissociate the complex and release the RNA. RNA was analysed on 1% agarose gel stained with GelGreen nucleic acid stain and visualized by ChemiDoc™ MP Imaging System (Bio-Rad). The RNA samples were quantified using a NanoDrop spectrophotometer before incubation with RNAse A. The protection of the RNA was determined by comparing the RNA bands after RNAse A treatment and dissociation of the complex to the free RNA in absence or presence of RNAse A.

Optical measurements and FRET analysis

ADGN-ava peptide labelled with Cyanin 5 (Cy5) was obtained from GENEPEP and sgRNA labelled with Cyanin 3 (Cy3) was obtained from IDT (Integrated DNA Technology). To prepare FRET donor-acceptor pairs, ADGN-PBN were prepared with Cy5 labelled ADGNava peptide, Cy3 labelled sgRNA and Cas9 mRNA in a 1:1 peptide to RNA weight ratio. The mixed solution was incubated for 30 minutes at room temperature in the dark.

Fluorescence spectra of labelled ADGN-PBN were recorded on the CLARIOstar Plus spectrometer (BMG Labtech), the excitation wavelength was set to 496 nm and the emission was collected between 525 and 738 nm. The FRET efficiency was calculated using the following equation:

E = (FDA -FA) / FDA
Where E is the FRET efficiency, FDA is the fluorescence intensity of the acceptor in the presence of the donor while FA term represents the fluorescence intensity of the acceptor in absence of the donor molecules. The fluorescence intensity of the donor and acceptor channels were measured from the fluorescence spectra.

Cell imaging by confocal microscopy

Fluorescently labelled sgRNA conjugated with Cy3 and unlabelled Cas9 mRNA were complexed with Cy5 labelled ADGN-ava peptide and non labelled ADGN-peg2, producing labeled PBN complexes with a peptide:RNA weight ratio of 1:1. A549-luc cells were plated in 4-well German borosilicate Lab-Tek coverslip chambers at a density of 30000 cells/well one day before transfection. Cells were transfected with 200 µl of ADGN-PBN in RPMI without phenol red (2 µg Cas9 mRNA and 2 µg of sgRNA per condition) as described above. 30 minutes after transfection, cells were stained with CellMask green actin stain (Invitrogen) 1X concentration as recommended by the supplier, at 37°C for 30 minutes. Live cells were then observed on 37°C pre-warmed chamber with CO2 by rapid confocal microscopy (Spinning disk) 1, 2, and 3 hours post transfection. A 63X oil immersion objective was used. Analysis was performed with ImageJ software.

Flow cytometry

One day before transfection, A549-luc cells were plated in 96-well plates at a density of 12500 cells/well in complete RPMI medium. Cells were transfected as described above (1µg ADGN complexed with 0.5 µg of Cas9 mRNA and 0.5 µg of sgRNA) and incubated for 3 hours. Cells were then washed with PBS 1X and trypsinized. After two more washes in PBS1X, the cell suspension (500 000 cells/Eppendorf) was passed through LSR™ II flow cytometer and data were acquired by using BD FACS Diva software (BD Biosciences). To discriminate between viable and dead cells and to exclude doublets, cells were appropriately gated by forward/size scatter. The results are expressed as percentage of gated cells which interacted with ADGN-PBN based on the experimental control (nontreated cells).

Luciferase activity measurement

A549-luc cells were plated in 96 well plates at a density of 12500 cells per well one day before transfection, as described above. 48 hours after transfection, the luciferase activity was evaluated using the luciferase assay system from Promega, according to the manufacturer's instructions. Briefly, cells were rinsed with PBS 1X, lysed with 20 µl of lysis buffer and incubated in 100 µl of luciferase detection reagent. The luciferase activity of the cell extracts was determined by the luminometric method described in the commercial protocol using a Clariostar Plus microplate reader (BMG Labtech). Results were normalized to luciferase activity of non-treated cells and cell viability or protein concentration of each sample. Statistical analysis was performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD). Student's t-test was used to compare the means of two groups, and one-way analysis of variance (ANOVA) was used to compare the means of multiple groups. A p-value of less than 0.05 was considered statistically significant.

Detection of mutation by T7E1 endonuclease assay

A T7 endonuclease assay was used to semi-quantitatively screen for gene editing efficiency. T7 endonuclease detects any structural deformities in the DNA heteroduplexes due to DNA mutations created by Cas9-induced dsDNA break and NHEJ repair (e.g., mismatches/insertions/deletions) and cleaves at that site. Thus, with higher gene editing efficiencies on the cell population, there are higher levels of heteroduplex formation and more product will be cleaved by T7 endonuclease. The T7 endonucleclease I (T7E1) was used to detect CRISPR-Cas9 genome editing events in targeted cells. GeneArt™ Genomic Cleavage Detection Kit (ThermoFisher) was used according to the manufacturer protocol. Briefly, genomic DNA was extracted at three days post-transfection of A549-luc with ADGN-PBN encapsulating CRISPR-Cas9 RNA components. Genomic target locus was PCRamplified using primers flanking the targeted sgDNA, shown in Table 12. The PCR products were then denatured and re-annealed by gradual cooling to produce potential heteroduplexes of target and mutated DNA strands where double-strand breaks have occurred. The DNA was then incubated at 37°C for 1 hour with the T7E1 enzyme, which recognizes the DNA mismatches and digest them. The resulting cleavaged and full-length DNA products were visualized on a 2% agarose gel electrophoresis. Gene editing efficiency was calculated using the equation below:

%𝑁𝐻𝐸𝐽 𝑒𝑣𝑒𝑛𝑡𝑠 = 1 -[(1 -𝑓𝑟𝑎𝑛𝑐𝑡𝑖𝑜𝑛 𝑐𝑙𝑒𝑎𝑣𝑒𝑑)] 1/2

Sequencing analysis

After transfection of cells with ADGN-PBNs encapsulating CRISPR-Cas9 RNAs, clonal cells were diluted to have a final concertation of 1 cell/well, seeded into 96-well plates, and allowed to grow for 2 weeks, or until the appearance of rounded colonies. Single colonies were harvested using trypsin and DNA was extracted using DNeasy blood and tissue kit from QIAGEN. PCR amplification was performed using a primer set designed to amplify the luciferase gene containing the sequence targeted by Luc gRNA. The PCR products were then purified using QIAquick PCR purification kit from Qiagen and sequenced using the Sanger method. The primer set used to amplify Luc sequence of interest are: 5'-GCAATCAAATCATTCCGGATAC-3' and 5'-GTCAATCAAGGCGTTGGTC-3'. Indels were analysed using TIDE (Tracking of Indels by Decomposition) analysis (https://tide.deskgen.com/). The analysis was done using non treated cells as reference sequence and the parameters were set to detect a maximum Indel size of 10 nucleotides.

Proliferation

Cells were cultures in opaque 96-well plates and transfected with ADGN-PBN as explained before. Cell proliferation was analyzed over a period of 5 days after transfection using the CellTiter Glow kit following manufacturer's instructions. Briefly, 100 µl of CellTiter-Glo® Reagent was added to the cells cultured in 100 µl cell culture medium. The content was mixed for 2 minutes on an orbital shaker to induce cell lysis.

The plate was incubated at room temperature for 10 minutes to stabilize luminescent signal and luminescence signal was recorded.

Western Blot

Western blotting was performed as previously described [START_REF] Busser | Amphiregulin Promotes BAX Inhibition and Resistance to Gefitinib in Non-small-cell Lung Cancers[END_REF]. 12,000-15,000 cells were planted in 96-well plate for 24 hours followed by ADGN-PBN treatment, which were indicated in the main text. The working medium volume was 200 µl /well. Samples from 8 wells were extracted and mixed together. For in vitro cells samples, 30 μg of protein were loaded onto SDS-polyacrylamide gel (10%). The list of antibodies used can be found on table The intensity of each band was measured by Vilber smart imaging system. Time-course exposure of proteins to chemiluminescence was used to perform semi-quantification of signal.

Trainings and congresses

Training courses I assisted to the following courses during my PhD studies:

-Jeudis de la sécurité : Le risque biologique.

-Jeudis de la sécurité : Le risque lié aux nanomatériaux.

-Formation de concepteur de projets d'expérimentation animale. CNRS.

-Face to face French courses for non-French speaking PhD students.

-Cours de français avec un enseignant à distance.

-Online French course for non-French speaking PhD students.

-Job-hunting strategies.

-My skill set portfolio: an invaluable stage in my PhD journey

Congress assisted

During my PhD I had the opportunity to share my work at:

-

List of tables

Table 1. Cas9 PAM sequences from different species. Adapted from [START_REF] Leonova | CRISPR/Cas9 technology in translational biomedicine[END_REF]. ________ 17 Table 2. Characteristics of different CRISPR-Cas9 delivery modes. Adapted from (L. [START_REF] Wang | Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy[END_REF]. ____ 33 Table 3. Overview of nanoparticles used for CRISPR-Cas9 delivery in the literature and their characteristics [START_REF] Duan | Nanoparticle Delivery of CRISPR/Cas9 for Genome Editing[END_REF]. ________________________________________________________________________________________________ 48 Table 4. Direct targeting of KRAS in cancer via the CRISPR systems. Adapted from [START_REF] Bender | CRISPR and KRAS: a match yet to be made[END_REF] (Kerschgens, n.d.). __ Figure 51. Luciferase knock-out evaluation 48 hours after transfection of A549-luc cells with ADGN-PBN. In the presence of my peers. With the completion of my doctorate in Biotechnology, in my quest for knowledge, I have carried out demanding research, demonstrated intellectual rigor, ethical reflection, and respect for the principles of research integrity. As I pursue my professional career, whatever my chosen field, I pledge to the greatest of my ability, to continue to maintain integrity in my relationship to knowledge, in my methods and in my results.

List of figures

Résumé

Le système CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) est apparu comme un outil révolutionnaire d'édition de gènes doté d'un énorme potentiel thérapeutique pour cibler directement les gènes à l'origine de diverses maladies telles que le cancer. KRAS est l'oncogène le plus fréquemment muté dans le cancer humain. Ses mutations G12D et G12V sont majoritaires dans l'initiation des mécanismes de carcinogénèse et se retrouvent notamment dans les cancers du pancréas et colorectal, Ces mutations restent des cibles cancéreuses les plus difficiles à atteindre. Le système CRISPR-Cas9 offre donc une opportunité intéressante de cibler ces mutations cancérigènes. Cependant, plusieurs défis doivent être relevés avant de pouvoir l'utiliser en clinique. L'un des facteurs clés est la méthode d'administration.

Outre le risque de dégradation de la nucléase et l'élimination rapide du système CRISPR-Cas9 par les macrophages, la grande taille de Cas9, la forte densité de charge anionique et la nature hydrophile de l'ARN constituent un obstacle important à la délivrance intracellulaire et à l'efficacité globale de la transfection des gènes.

Nous avons donc développé de nouvelles nanoparticules à base de peptides (PBN) qui constituent un système de vectorisation qui a été identifié comme l'une des stratégies non virales les plus prometteuses pour l'administration intracellulaire d'acides nucléiques. Nous avons démontré que ces PBN forment des complexes stables avec l'ARNm Cas9 et son ARNg et qu'elles sont capables de protéger les composants du système CRISPR-Cas9 de la dégradation par les nucléases. En utilisant la microscopie confocale à fluorescence, nous avons confirmé la délivrance intracellulaire du système. Nous avons d'abord vérifié que le système CRISPR-Cas9 délivré était fonctionnel en ciblant le gène rapporteur de la luciférase. Les PBN ont généré efficacement le knock-out du gène de la luciférase in vitro sans aucune cytotoxicité apparente. Les PBN ont permis d'éditer de manière robuste le gène de la luciférase avec une efficacité de 60 % et une diminution de 50 % de l'activité globale de celle-ci. De plus, nous avons montré que les PBN ont pu cibler les mutations prédominantes du gène KRAS, atteignant une efficacité de 20 % pour la mutation G12D et de 60 % pour la mutation G12V. La phosphorylation des principales protéines en aval des voies de signalisation de KRAS a été régulée à la baisse et le taux de prolifération cellulaire a été rétabli après l'édition CRISPR-Cas9.

Nous avons montré pour la première fois, que des nanoparticules à base de peptides étaient capables de délivrer dans des cellules cancéreuses le système CRISPR-Cas9 sous forme d'ARN et de cibler de manière efficace les mutations G12D et G12V de l'oncogène KRAS. Nos résultats soulignent l'importance d'un système d'administration approprié pour délivrer CRISPR-Cas9 et ouvrent des perspectives thérapeutiques prometteuses pour cibler les mutations de KRAS participant au développement du cancer.

Figure 1 .

 1 Figure 1. Schematic representation of endonucleases used for gene editing technologies. (A) The Meganuclease (MegN) called I-SceI mediates the DNA double strand break. (B) The zinc finger nuclease (ZFN) contains two different ZFN monomers fused with Flavobacterium okeanokoites (FokI) nuclease domain. Their binding to a nucleotide triplet results in dimerization of the FokI that allows DNA cleavage between ZFN recognition sites. (C) The transcription activator like effector nucleases (TALENs) are composed of two monomers with each containing a TALE DNA binding domain and a FokI nuclease domain. Fok1 dimerizes to create a double-strand break. (D) Clustered Regularly Interspaced Short Palindromic Repeats associated protein Cas (CRISPR-Cas) is a two-component system composed of Cas9 and a gRNA. Once Cas9 finds a PAM site, if the gRNA binds to the DNA, a double break occurs three base pairs upstream the PAM) (Premalatha & Mayanglambam, 2020).

Figure 4 .

 4 Figure 4. Timeline of the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system with important milestones (Nidhi et al., 2021).

Figure 5 .

 5 Figure 5. Domain organization for the type II spCas9 in its non-active and active form (A) Schematic illustration of spCas9 inactive form and surface representations of the three-dimensional structure of spCas9. The Cas9 protein is comprised of few domains: Recognition (REC), Bridge Helix (BH), RuvC, HNH, and PAM Interacting (PI) domains. (ID 4CMP from PDB database). (B) Schematic illustration of 3D structure of complex spCas9 active form with sgRNA and its surface representations (ID 4ZT0 from PDB database). (C) Schematic illustration of 3D structure of complex spCas9 active form with sgRNA and target DNA and its surface representations (ID 5Y36 from PDB database). (D) Cartoon representation of the spCas9-sgRNA-cut 3D structure of DNA (ID 5Y36 from PDB database). All crystal structural images were visualized with the PyMOL Molecular Graphics System. (Leonova & Gainetdinov, 2020).

Figure 6 .

 6 Figure 6. DNA repair mechanisms after CRISPR-Cas9 gene editing. CRIPSPR-Cas9 cleavage activity creates a double-strand break on the target DNA, which is repaired either by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Image adapted from (Dragt, 2020) Biorender.

Figure 8 .

 8 Figure 8. Major DNA repair mechanisms. The error-free Homologous directed repair requires a DNA template to repair the DNA DSB. It involves resection of the DNA ends by various nucleases. The ssDNA generated is then bound by RPA. Next, there is an exchange of RPA for RAD51, which facilitates the homology search and repair of the DSB. The non-homologous end joining repair machinery does not require a DNA template but generate errors in the DNA sequence. The DNA ends are bound by Ku70/80 heterodimers promoting the binding of DNA-PKcs and the recruitment of ARTEMIS. This creates a binding platform for XRCC4, XLF, and LIG4, which facilitate ligation of the DNA ends (Brochier & Langley, 2013).

Figure 9 .

 9 Figure9. Prevalent DNA repair pathway during the different stages of the cell cycle. Non-homologous end joining (NHEJ, in purple) is active during the whole cell cycle except in mitosis, homology-directed repair (HDR, in blue) is mainly active during the S/G2 phases and microhomology-mediated end joining (MMEJ, in yellow) is an alternative NHEJ pathway mainly active during the G1/early S phases. Adapted from[START_REF] Gallego | Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing[END_REF]

Figure 12 .

 12 Figure 12. Delivery methods for CRISPR-Cas9. The schematic illustrations represent varieties of the tools to deliver genome editing components. The delivery tools are roughly classified into three categories: biological, chemical, and physical methods, based on the constituents and cellular entry mechanisms.Adapted from[START_REF] Taha | Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges[END_REF].

Figure 15 .

 15 Figure 15. Sonoporation physical method to deliver CRISPR-Cas9. (A) Schematics of sonoporation mechanism (Fajrial et al., 2020). (B) RFP and GFP expression in Hela cells after acoustic transfection of CRISPR-Cas9 using AAV-LifeAct-TagRFP and AAV-CAGGS-EGFP DNA templates for HDR. Scale bars indicate 20 µ (Yoon et al., 2017).

Figure 16 .

 16 Figure 16. Viral vectors for CRISPR-Cas9 delivery. Main virus delivery strategy involve lentivirus (LV), adenovirus (AdV), adeno-associated virus (AAV) and virus like particles (VLP). Adapted from[START_REF] Moscoso | The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases[END_REF]

Figure 18 .

 18 Figure18. Critical obstacles for nanoparticle mediated delivery of CRISPR-Cas9. When administered systemically, NPs must protect their cargo from destabilizing serum components, enzyme degradation, and clearance by immune cells or liver and kidneys clearance. To facilitate efficient intracellular uptake, the protein or nucleic acid cargo should be (1) encapsulated within nanocarriers. Upon endocytosis into the cell, the nanoparticles must (2) escape from degradative endolysosomal compartments. In the case of mRNA delivery, cargo release must occur in the cytosol (4a) to enable translation by ribosomes (5a). In DNA delivery, plasmid DNA needs to be transported to the nucleus (4b) for transcription into mRNA (5b), which must then enter the cytosol to undergo translation into protein (6b). Protein complexes, whether synthesized within the cell (6a, 7b) or delivered directly by the nanocarrier (4c), must localize to the nucleus for gene editing to take place. The common intracellular delivery steps applicable to all cargo formats are represented by black arrows, while red, green, and blue arrows indicate steps specific to mRNA, plasmid DNA, and protein complexes, respectively[START_REF] Rui | Non-Viral Delivery To Enable Genome Editing[END_REF].

Figure 20 .

 20 Figure 20. Schematic diagram of cationic-lipid assisted PLGA nanoparticles (CLAN) induced macrophages targeted gene disruption. The CRISPR/Cas9 plasmid contained cell-specific promotor-human CD68promoter, thus is specifically expressed in macrophages. After intravenous injection in mouse, nanoparticles could be endocytosed by diverse cells, including tissue cells, T cells, B cells, neutrophils, monocytes, and macrophages. However, the CD68 promoter ensured the Cas9 expression occurred only in monocytes and macrophages and led to gene knockdown[START_REF] Luo | Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles[END_REF].

Figure 21 .

 21 Figure 21. Design of SPPF-Dex (Dexamethasone)nanoparticles for CRISPR/Cas9 delivery, and illustration of the intracellular genome editing process upon 808 nm laser irradiation. Upon the NIR-II laser irradiation, the distribution of SPPF-Dex can be tracked in vivo. Simultaneously, the photothermal conversion of SPPF facilitates the endolysosomal escape of SPPF-Dex and release of CRISPR-Cas9. Dex dilates the nuclear pores to initiate the translocation of CRISPR-Cas9 for genome editing. (L. Li et al., 2019) (L. Li et al., 2019).

Figure 22 .

 22 Figure22. CRISPR-Gold NPs deliver Cas9 ribonucleoprotein and donor DNA and induce homology directed DNA repair. A) CRISPR-Gold is composed of 15 nm GNPs conjugated to thiol-modified oligonucleotides (DNA-Thiol), which are hybridized with single-stranded donor oligonucleotides and subsequently complexed with Cas9 RNP and the endosomal disruptive polymer PAsp(DET), where 'DET' is diethylenetriamine.B) CRISPR-Gold is injected into the hind leg muscle of mdx mouse and allows the correction of dystrophin mutations after 2 weeks (K.Lee et al., 2018).

Figure 23 .

 23 Figure 23. Formulation of peptide-based nanoparticles in the presence of different nucleic acids and their cellular internalization. Peptide-based nanoparticles (PBNs) are formulated by mixing a cell-penetrating peptide (CPP) or a grafted CPP (PEGylated, targeting sequence or fatty acid) with a nucleic acid (NA) such as pDNA, mRNA, siRNA, or ASO at a given molar or charge ratio. By mixing these two compounds, the nanoparticle is formed by self-assembling into naked PBNs (a), a multi-grafted PBNs (b), or prospective micelle-like PBNs (no model available) (c).In all cases, the PBNs of mean size between 60 nm and 150 nm encapsulate several NAs for cellular transfection. Thereafter, cellular internalization could occur via direct translocation (d) or via endocytosis-dependent pathways (f). After the direct translocation (e) or endosomal escape (g), the NAs could be active either by silencing or activating genes or by performing splice modulation (h). GAG = glycosaminoglycans[START_REF] Boisguérin | Peptide-based nanoparticles for therapeutic nucleic acid delivery[END_REF].

Figure 24 .

 24 Figure 24. Schematic illustration showing the formation of P-HNPs after the interaction of helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)amino-methyl)benzyl-L-glutamate) (PPABLG), PEG2k-T40 peptide and the Cas9/sgRNA plasmid. Cellular internalization of the PBN by endocytosis or cellular pore and intracellular activity of Cas9/sgRNA plasmid to perform genome editing (H. X. Wang et al., 2018).

Figure 25 .

 25 Figure25. Timeline of KRAS discovery[START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

Figure 27 .

 27 Figure 27. KRAS signaling pathway with main proteins involved. Receptors in the EGFR family upstream of KRAS transmit extracellular signals to activate KRAS. KRAS function is controlled by cycling through an inactive, GDP-bound state and an active, GTP-bound state. Activation of KRAS occurs when it binds to Guanine Nucleotide Exchange Factor (GEF) proteins, which initiate the exchange of GDP for GTP. One of the GEFs, SOS1, is a RAS activator. Active, GTP-bound KRAS can trigger activation of downstream signaling molecules including RAF/MEK/ERK, PI3K/AKT/mTOR, and RAL/NF-κβ pathways, which result in the expression of genes that promote cellular survival, proliferation and differentiation. It can self-inactivate by cleaving the terminal phosphate group on the GTP, converting it to GDP. This inactivation process is accelerated by GTPase-activating (GAP) proteins. Adapted from[START_REF] Fakih | Drugging KRAS : current perspectives and state -of -art review[END_REF].

Figure 28 .

 28 Figure 28. Distribution of KRAS alleles/amplification and patient numbers in cancer. Mutation and amplification rates for KRAS have been derived from the AACR GENIE 9.0-public database (Sweeney et al., 2017), whereas new tumor type-specific diagnoses have been extracted from ACS Cancer Facts and Figures 2020 (Hallquist Viale, RN, MS, CNS, ANP, 2020), scaling the cases of lung adenocarcinoma to 40% of all lung cancers. A total of 81,996 samples with mutation and copy number profiles were collapsed into individual patient samples and filtered for distinct alleles and amplification in KRAS. Notably, alleles not covered in the top ranks were collapsed into the class 'other'. The grouping 'multiple' contains all cases, for which different KRAS alterations have been observed in a single patient, for example, two different mutations or a mutation coupled with KRAS amplification. (A) Distribution of KRAS mutations/amplification across 11,956 patients and tumor types. (B) Absolute new diagnoses/patient numbers of new KRAS-mutated cases split by tumor type and allele/alteration. (C) KRAS alterations across all cases of lung adenocarcinoma (n = 7444), colorectal cancer (n = 5751), and pancreatic ductal adenocarcinoma (n = 2796) in the AACR GENIE database (Herdeis et al., 2021).

Figure 29 .

 29 Figure 29. Schematic representation of evolution of ductal epithelial cells into Pancreatic Ductal Adenocarcinoma (PDAC). Involvement of driver mutations in the progression of lung cancer pathogenesis.An oncogenic KRAS mutation is the initiator of tumorigenic progression in lung cancer, causing hyperplasia to develop into adenoma. Mutations in CDNK2A, BRCA1/2, p53 and SMAD4 contribute to carcinogenic switching of the tissue. (Darkening of the colors in the cells indicate progression of cancer stage)[START_REF] Bender | CRISPR and KRAS: a match yet to be made[END_REF].

Figure 35 .

 35 Figure 35. Strategy used for ADGN-PBN mediated CRISPR-Cas9 gene editing of target gene. ADGN-PBN encapsulate Cas9 mRNA and sgRNA and mediates its cellular internalization and delivery into the cytosol.Once released, Cas9 mRNA is translated into the Cas9 protein thar forms the RNP complex and penetrates the cell nucleus. CRISPR machinery scans the DNA and creates a DNA DSB when finds a complementary sequence to the sgRNA next to a protospacer adjacent motif (PAM). The NHEJ, an error prone DNA repair mechanism, is activated by the cell leading to introduction of indels in the DNA and therefore knock out of the gene.

Figure 36 .

 36 Figure 36. Schematic representation of the structural conformation of ADGN-ava (A) and ADGN-peg2 (B) peptides analysed with PyMOL Molecular Graphics System.

Figure 37 .

 37 Figure 37. Chemical structure of 5-aminovaleric acid (Ava) linker (A) and polyethylene glycol molecule (B) used for ADGN-Ava and AGDN-peg2 synthesis respectively.

Figure 38 .

 38 Figure 38. Encapsulation of fLuc mRNA by ADGN peptides. (A) Encapsulation efficiency evaluated by agarose gel electrophoresis after GelGreen staining. 0.5 μg of fLuc mRNA were complexed with ADGN peptides in a peptide:RNA weight ratio of 0.5:1, 1:1, 2:1, 3:1 and 5:1 and (B) quantification of RNA complexation for each condition.

Figure 40 .

 40 Figure40. Luciferase expression levels on Panc-1 cells after transfection with ADGN-PBN complexed with 0.8 µg fLuc mRNA at increasing peptide:RNA weight ratios (2:1, 3:1, 5:1 and 10:1). Luciferase activity was measured with the luciferase assay system 48 hours post-transfection. Results are presented as mean ± SEM. n= 3 independent experiments performed in triplicates.

Figure 41 .

 41 Figure 41. Luciferase expression levels on different cell lines after transfection with ADGN-PBN complexed with 0.8 µg fLuc mRNA and 2:1 peptide:RNA weight ratio. Luciferase activity was measured with the luciferase assay system 48 hours post-transfection. Results are averages of 3 independent experiments performed in triplicates and are presented as mean ± SEM. Statistical analysis was performed using a onetailed non-parametric t test. P-value < 0.05.

Figure 42 .

 42 Figure 42. Encapsulation of CRISPR-Cas9 RNA components by ADGN peptides. (A) Encapsulation efficiency evaluated by agarose gel electrophoresis. 0.5 μg of Cas9 mRNA and 0.5 μg of sgRNA were complexed with ADGN peptides in a peptide:RNA weight ratio of 0.25:1, 0.5:1, 1:1 and 2:1 and (B) quantification of the percentage of RNA complexation for each condition. Samples were run on 0.8 % agarose gel and stained with GelGreen nucleic acid stain.

Figure 43 .

 43 Figure 43. Size distribution of ADGN-PBNs complexed with CRISPR-Cas9 RNA components in a 1:1 peptide:RNA weight ratio. (A) Representative DLS intensity-based size distribution histogram and (B) Transmission electron microscopy (TEM).

Figure 45 .

 45 Figure 45. Cellular interaction of CRISPR-Cas9 system encapsulated in ADGN-PBN by A549 cells. Flow cytometry analysis of Cy5-lalelled ADGN-ava peptide and Cy3-labelled sgRNA interactions with live A549 cells after 3 hours of incubation.

Figure 46 ,

 46 Figure46, ADGN-PBNs are detected at the periphery of the cell and in the cytoplasm after 1 hour of incubation with the cancer cells. The low level of spots corresponding to Cy3labelled sgRNA suggests that Cy3 fluorescence may not be detected because of sgRNA are entrapped into the nanoparticle whereas Cy5 labelled peptide are at the surface of the nanoparticles creating a quenching of the Cy3 signal. It is to be noted that to validate this hypothesis, further experiments such as Forster resonance energy transfer (FRET) and Fluorescence lifetime imaging microscopy (FLIM) should be performed. After 2 hours, homogeneous distribution of the complex is observed in the cytoplasm and no clear accumulation at a precise region can be distinguished. Colocalizations between sgRNA and the peptide appear as well as individual sgRNA and peptide spots suggesting a partial dissociation of the cargo from the nanocages. No significant alternation in cell morphology was observed in cells treated with ADGN-PBN. This result indicated that ADGN-PBN are not just tightly adsorbed to the cell surface, but indeed being internalized into the cells and delivering its RNA cargo to the cells.

Figure 46 .

 46 Figure 46. Cellular uptake of ADGN-PBN encapsulating CRISPR-Cas9 system by A549 cells. Cells were transfected with ADGN-PBN containing Cy5-labelled ADGN peptide (red color), 0.5 μg of Cas9 mRNA and 0.5 μg of Cy3-labelled sgRNA (green color) in a 1:1 peptide:RNA weight ratio. Cell Mask was used to stain the cells. The cellular uptake was monitored by confocal fluorescence microscopy 1, 2 and 3 hours posttransfection (scale bar: 10 μm).

  Cy3 and Cy5 fluorophores have suitable spectral overlap shown on Figure47. When the Cy5 labelled ADGN peptide and Cy3 labelled sgRNA are complexed, these donor and acceptor fluorophores are in close physical proximity. Under these conditions, the emission band of the donor molecule overlaps with the excitation band of the energy acceptor molecule and in consequence, the energy from a photon absorbed by the donor cyanin 3 molecule is transferred to the cyanin 5 acceptor molecule causing the later to fluoresce as if it was exited directly (see Figure47). As a result of FRET, the donor emission is reduced while the acceptor emission increases[START_REF] Ishikawa-Ankerhold | Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM[END_REF].

Figure 47 .

 47 Figure 47. Overlap between the Cy3 emission spectrum and the excitation spectrum of Cy5. As a result of FRET, the donor emission (Dem) is reduced while the acceptor emission (Aem) increases (Ishikawa-Ankerhold et al., 2012).

Figure 48 .

 48 Figure 48. Fluorescence spectrum of ADGN-PBN containing Cy5-labelled ADGN-ava complexed with Cy3labelled sgRNA and Cas9 mRNA at 1:1 or 0.5:1 peptide:RNA weight ratio compared to ADGN-PBN containing Cy5-labelled ADGN-ava peptide and non-labelled RNAs. Excitation wavelength: 496 nm.

Figure 49 .

 49 Figure 49. Efficient in vitro luciferase downregulation in cells transfected with CRISPR-Cas9 delivered by ADGN-PBN. Normalized luciferase activity in luciferase expressing A549 (A) or MCF7 cell lines (B) was

  .

Figure 50 .

 50 Figure 50. Gene editing detection by of T7-endonuclease I (T7E1) assay. (A) Schematics of the methodology and the analysis of gene editing mutation detection T7E1 assay. (B) Representative gel images of results of T7E1 assay. Cells were transfected with the CRISPR-Cas9 system delivered by ADGN-PBN at a 1:1 peptide to RNA weight ratio or the control delivery reagent polyplus JetMessenger. Adapted from(Kerschgens, n.d.).

Figure 51 .

 51 Figure 51. Luciferase knock-out evaluation 48 hours after transfection of A549-luc cells with ADGN-PBN complexed with 0.5 ug Cas9 mRNA and 0.5 ug sgRNA at a peptide to RNA weight ratio of 1:1. (A)Schematic representation of qPCR based mutation detection method. (B) qPCR amplification results. Results are averages of 3 independent experiments and are presented as mean ± SEM. Statistical analysis was performed using ANOVA one-way analysis of variance followed by Dunnett's test. *Indicates p < 0.05 between groups. **Indicates p < 0.01 vs. non treated cells.

Figure 52 .

 52 Figure 52. Schematics of Cas9 protein and sgRNA Luc targeting the luciferase gene. PAM sequence is shown in yellow, targeted DNA strand is shown in blue, sgRNA Luc is shown in red and the expected break site is shown as a vertical dotted line. Adapted from (Straume et al., 2020).

Figure 53 .

 53 Figure 53. Sanger sequencing analysis of CRISPR-Cas9 edited A549-Luc cells. (A) Representative chromatograms of wild type A549-luc cells and CRISPR-Cas9 edited cells after transfection with ADGN-PBN. (B) Indels profile observed after treatment. sgRNA and PAM are shown in grey and yellow respectively. The expected cutting site is shown with a vertical dotted line. Indels (in red) occur 4bp from the cutting site.

Figure 54 .

 54 Figure 54. Tracking of indels by decomposition (TIDE) analysis. (A) Representative visualization of aberrant sequence signal in wild type (black) and CRISPR-Cas9 edited sample (green), the region used for decomposition is shown with a grey bar. (B) Representative decomposition yielding the spectrum of indels and their frequencies.

Figure 55 .

 55 Figure 55. Schematic representation of the in vivo orthotopic lung cancer model. This figure illustrates the experimental design and timeline of the in vivo model used.Luciferase-expressing A549 cells were orthotopically implanted into the lungs of nude mice. Following tumor engraftment, the mice were allowed to harbor the tumors for a period of 16 days to allow for substantial growth. Subsequently, ADGN-PBN carrying the CRISPR-Cas9 system were administered intravenously. The mice were then monitored for an additional 72 hours to assess the in vivo bioluminescence levels as a measure of response to the treatment and gene editing activity.

Figure 56 .

 56 Figure 56. In vivo efficiency of luciferase targeting CRISPR-Cas9 system delivered by ADGN-PBN. In vivo bioluminescence imaging and quantification of mice with orthotopic lung tumor, before and 72h after the intravenous injection of ADGN-PBN complexed with CRISPR-Cas9 with a molar ratio of 20 or 200.

Figure 57 .

 57 Figure 57. Schematic representation of the therapeutic strategy targeting the predominant KRAS mutations by ADGN-PBN complexed with CRISPR-Cas9 system.

Figure 59 .

 59 Figure 59. Mutant KRAS knock-out evaluation by qPCR-based mutation detection method. Relative qPCR quantification 48 hours after transfection of (A) Panc-1 cells and (B) SW480 cells with ADGN-PBN complexed with 0.5 µg Cas9 mRNA and 0.5 µg sgRNA targeting the specific mutation at a peptide to RNA weight ratio of 1:1. Mutation specific amplification curves of G12D KRAS (C) and G12V KRAS (D). Results are normalized with the amplification of the internal control primer set and are presented as mean ± SEM, n=3 independent experiments performed in triplicates. Statistical analysis was performed using ANOVA one-way analysis of variance followed by Dunnett's test. *Indicates p < 0.05 between groups. **Indicates p < 0.01 vs. non treated cells.

Figure 60 .

 60 Figure 60. Mutated KRAS expression levels in PANC-1 and SW480 cell lines. Representative Western Blots (A) and quantification (C) of G12V mutated KRAS expression levels in Panc-1 cells. Representative Western Blots (B) and quantification (D) of G12V mutated KRAS expression levels in SW480 cells. Protein extracts from both cell lines were analyzed 48 hours after transfection with ADGN-PBN complexed with 0.5 µg Cas9 mRNA and 0.5 µg sgRNA (12V1, 12V2, 12V3, 12D1, 12D2 or 12D3) at 1:1 peptide to RNA weight ratio. GAPDH was used as a loading control. Values are mean ± SEM, n=2. Statistical analysis was performed using ANOVA one-way analysis of variance followed by Dunnett's test. *Indicates p < 0.05 vs. non treated cells. **Indicates p < 0.01 vs. non treated cells.

Figure 61 .

 61 Figure 61. Representative westerb blots of the expression levels of pAKT, AKT, pERK and ERK proteins on (A) Panc-1 cells or (B) SW480 cells 48 hours after transfection with CRISPR-Cas9 system delivered by ADGN-PBN. 0.5 µg Cas9 mRNA, 0.5 µg sgRNA and 1:1 peptide:RNA ratio were used. SgRNA12D3 and 12V3 were used for each cell line respectively. Here the prefix "p" indicates the phosphorilated version of the protein. GAPDH was used as a loading control.

Figure 62 .

 62 Figure 62. Western blot quantification. AKT, pAKT, ERK and pERK expression levels after ADGN-PBN mediated CRISPR-Cas9 editing of G12D mutant KRAS in Panc-1 cells (n=2).

Figure 63 .

 63 Figure 63. Western blot quantification. AKT, pAKT, ERK and pERK expression levels after ADGN-PBN mediated CRISPR-Cas9 editing of G12V mutant KRAS in SW480 cells (n=2).

Figure 64 .

 64 Figure 64. Cell proliferation assay. CellTiter Glow system was used to evaluate cell proliferation 5 days post transfection of (A) Panc-1 cells and (B) SW480 cells with ADGN-PBN complexed with 0.5 µg Cas9 mRNA and 5 µg sgRNA with 1:1 peptide:RNA weight ratio. sgRNA 12D2 and 12D3 were used to target G12D KRAS mutation while sgRNA 12V2 and 12V3 were used to target G12V KRAS mutation. n=3

Figure 65 .

 65 Figure 65. Schematic representation of the experimental design and timeline of the in vivo xenografted pancreatic or colorectal cancer models. This diagram outlines the experimental design for xenograft models involving subcutaneous implantation of human pancreatic cancer cells (Panc-1) and colorectal cancer cells (SW403) in female Balb/C nude mice. After reaching a tumor size of 100 mm 3 , mice were randomly divided into four groups for treatment with ADGN-PBN, delivering CRISPR-Cas9 RNA targeting G12D or G12V KRAS mutations. Injections were administered intravenously on days 1 and 7 at doses of 0.5 mg/kg or 1 mg/kg. Tumor dimensions were monitored at 5-day intervals.

Figure 66 .

 66 Figure 66. In vivo evaluation of ADGN-PBN targeting G12D KRAS mutation. Female Balb/C nude mice were xenographted subcutaneously with Panc-1 cells. (A) Tumor size and (B) body weight after ADGN-PBN intravenous administration at day 1 and 7. Saline and ADGN-PBN encapsulating sgRNA G12C were used as controls and administered i.v. n=5.

Figure 67 .

 67 Figure 67. In vivo evaluation of ADGN-PBN targeting G12V KRAS mutation. Female Balb/C nude mice were xenographted subcutaneously with Panc-1 cells. (A) Tumor size and (B) body weight after ADGN-PBN intravenous administration at day 1 and 7. Saline and ADGN-PBN encapsulating sgRNA G12C were used as controls and administered i.v. n=5.

  SFnano 2021 annual meeting. Oral presentation. Anger, France. -Oncoriales 2021. Cancéropôle Lyon Auvergne Rhône-Alpes. Lyon, France. -SFnano 2022 annual meeting. Poster presentation. Strasbourg, France. -2022 CRISPR Screening in Cancer Discovery meeting. Marseille, France. -EARC European association of cancer research congress 2023. Poster presentation. Torino, Italy. Prices -2021 SFnano image contest. First Price.

Figure 69 .

 69 Figure 69. qPCR standard curves. Genomic DNA from A549 cells stably expressing luciferase was serially diluted four times to generate the regression curves for qPCR relative quantification after ADGN-PBN treatment. Primers IN (A) the luciferase target point and internal control primers OUT (B) outside of the expected Cas9 cutting site serve for normalization purpose.

Figure 70 .B

 70 Figure 70. Melting peaks of qPCR amplified samples using (A) KRAS G12D primers or (B) primers control.

Figure 1 .

 1 Figure 1. Schematic representation of endonucleases used for gene editing technologies. ____________________ Figure 2. Streptococcus' adaptive immune system against viruses: CRISPR-Cas9. _____________________________ Figure 3. Conventional classification of CRISPR-Cas systems[START_REF] Gostimskaya | CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing[END_REF]. ___________________________ Figure 4. Timeline of the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system. _____ Figure 5. Domain organization for the type II spCas9 in its non-active and active form. ______________________ Figure 6. DNA repair mechanisms after CRISPR-Cas9 gene editing. ____________________________________________ Figure 7. Early steps of DNA damage response. _________________________________________________________________ Figure 8. Major DNA repair mechanisms. ________________________________________________________________________ Figure 9. Prevalent DNA repair pathway during the different stages of the cell cycle. ________________________ Figure 10. Challenges and overcoming strategies of CRISPR/Cas9. ____________________________________________ Figure 11. CRISPR/Cas9 system delivered using pDNA, mRNA or RNP. ________________________________________ Figure 12. Delivery methods for CRISPR-Cas9. ___________________________________________________________________ Figure 13. Electroporation delivery of CRISPR-Cas9 RNP components. ________________________________________ Figure 14. Physical delivery of CRISPR-Cas9 by microinjection. ________________________________________________ Figure 15. Sonoporation physical method to deliver CRISPR-Cas9. _____________________________________________ Figure 16. Viral vectors for CRISPR-Cas9 delivery. ______________________________________________________________ Figure 17. Nanoscale integration of biomolecules and main nanoparticles types._____________________________ Figure 18. Critical obstacles for nanoparticle mediated delivery of CRISPR-Cas9. _____________________________ Figure 19. Important characteristics of nanoparticles as delivery systems. ____________________________________ Figure 20. Schematic diagram of cationic-lipid assisted PLGA nanoparticles (CLAN) induced macrophages targeted gene disruption. _________________________________________________________________________________________ Figure 21. Design of SPPF-Dex (Dexamethasone)nanoparticles for CRISPR/Cas9 delivery. ___________________ Figure 22. CRISPR-Gold NPs deliver Cas9 ribonucleoprotein. ___________________________________________________ Figure 23. Formulation of peptide-based nanoparticles in the presence of different nucleic acids and their cellular internalization. ___________________________________________________________________________________________ Figure 24. Schematic illustration showing the formation of P-HNPs. __________________________________________ Figure 25. Timeline of KRAS discovery. __________________________________________________________________________ Figure 26. KRAS gene and mutational hotspot. __________________________________________________________________ Figure 27. KRAS signaling pathway with main proteins involved. ______________________________________________ Figure 28. Distribution of KRAS alleles/amplification and patient numbers in cancer. _______________________ Figure 29. Schematic representation of evolution of ductal epithelial cells into Pancreatic Ductal Adenocarcinoma (PDAC). _________________________________________________________________________________________ Figure 30. Schematic representation of CRC development upon occurrence of mutations. ___________________ Figure 31. Schematic representation of driver mutations in the advancement of lung cancer. _______________ Figure 32. KRAS signaling pathway with main targeting inhibitors . __________________________________________ Figure 33. KRAS G12C protein structure with switch II containing adagrasib or sotorasib inhibitors. _______ Figure 34. Resistance mechanisms to sotorasib and adagrasib. ________________________________________________ Figure 35. Strategy used for ADGN-PBN mediated CRISPR-Cas9 gene editing of target gene. ________________ Figure 36. Schematic representation of the structural conformation of ADGN-ava (A) and ADGN-peg2 (B) peptides analysed with PyMOL Molecular Graphics System. ____________________________________________________ Figure 37. Chemical structure of 5-aminovaleric acid (Ava) linker (A) and polyethylene glycol molecule (B) used for ADGN-Ava and AGDN-peg2 synthesis respectively. ____________________________________________________ Figure 38. Encapsulation of fLuc mRNA by ADGN peptides. ____________________________________________________ Figure 39. Representative DLS intensity-based size distribution histogram. ___________________________________ Figure 40. Luciferase expression levels on Panc-1 cells after transfection with ADGN-PBN. __________________ Figure 41. Luciferase expression levels on different cell lines after transfection with ADGN-PBN.____________ Figure 42. Encapsulation of CRISPR-Cas9 RNA components by ADGN peptides. _______________________________ Figure 43. Size distribution of ADGN-PBNs complexed with CRISPR-Cas9 RNA. _______________________________ Figure 44. Efficient RNA protection by ADGN-ava from RNAse A degradation. ________________________________ Figure 45. Cellular interaction of CRISPR-Cas9 system encapsulated in ADGN-PBN by A549 cells.. __________ Figure 46. Cellular uptake of ADGN-PBN encapsulating CRISPR-Cas9 system by A549 cells. _________________

Figure 52 .

 52 Figure 47. Overlap between the Cy3 emission spectrum and the excitation spectrum of Cy5. _________________ Figure 48. Fluorescence spectrum of ADGN-PBN containing Cy5-labelled ADGN-ava complexed with Cy3labelled sgRNA and Cas9 mRNA. _________________________________________________________________________________ Figure 49. Efficient in vitro luciferase downregulation in cells transfected with CRISPR-Cas9 delivered by ADGN-PBN. ________________________________________________________________________________________________________ Figure 50. Gene editing detection by of T7-endonuclease I (T7E1) assay. dapted from(Kerschgens, n.d.). __ Figure51. Luciferase knock-out evaluation 48 hours after transfection of A549-luc cells with ADGN-PBN. Figure 52. Schematics of Cas9 protein and sgRNA Luc targeting the luciferase gene. ________________________ Figure 53. Sanger sequencing analysis of CRISPR-Cas9 edited A549-Luc cells. _______________________________ Figure 54. Tracking of indels by decomposition (TIDE) analysis. ______________________________________________ Figure 55. Schematic representation of the in vivo orthotopic lung cancer model. ___________________________ Figure 56. In vivo efficiency of luciferase targeting CRISPR-Cas9 system delivered by ADGN-PBN. __________ Figure 57. Schematic representation of the therapeutic strategy targeting the predominant KRAS mutations by ADGN-PBN complexed with CRISPR-Cas9 system. ___________________________________________________________ Figure 58. Schematics of the methodology employed for the qPCR-based mutation detection method. _____ Figure 59. Mutant KRAS knock-out evaluation by qPCR-based mutation detection method. ________________ Figure 60. Mutated KRAS expression levels in PANC-1 and SW480 cell lines. _________________________________ Figure 61. Representative westerb blots of the expression levels of pAKT, AKT, pERK and ERK proteins on (A) Panc-1 cells or (B) SW480 cells. _________________________________________________________________________________ Figure 62. Western blot quantification. AKT, pAKT, ERK and pERK expression levels in Panc-1. ____________ Figure 63. Western blot quantification. AKT, pAKT, ERK and pERK expression levels in SW480. ____________ Figure 64. Cell proliferation assay. ______________________________________________________________________________ Figure 65. Schematic representation of the experimental design and timeline of the in vivo xenografted pancreatic or colorectal cancer models. ________________________________________________________________________ Figure 66. In vivo evaluation of ADGN-PBN targeting G12D KRAS mutation. ________________________________ Figure 67. In vivo evaluation of ADGN-PBN targeting G12V KRAS mutation. ________________________________ Figure 68. Melting peaks of qPCR amplified samples . __________________________________________________________ Figure 69. qPCR standard curves. . ______________________________________________________________________________ Figure 70. Melting peaks of qPCR amplified samples. __________________________________________________________ Figure 71. Melting peaks of qPCR amplified samples primers control. ________________________________________ Figure 72. qPCR standard curves. _______________________________________________________________________________

Aims of the project ________________________________________ Results part 1: ADGN-PBN are an efficient mRNA delivery system ___________________________________________

	1.1 Mechanistic insights into site-specific genome editing _______________________
	1.2 Tracing the bacterial origins of CRISPR-Cas ___________________________________
	1.3 Evolution of CRISPR-Cas9 in biotechnology: An overview ____________________
	1.4 Decoding CRISPR-Cas9: Components for precise gene editing _______________
	1.5 Navigating DNA double-strand break repair mechanisms ____________________
	1.6 Factors influencing DNA repair outcomes of Cas9-induced double-strand
	breaks _____________________________________________________________________________________
	1.7 Challenges associated to CRISPR-Cas9 delivery and gene editing____________
	1.8 Exploring diverse CRISPR-Cas9 delivery approaches _________________________
	2.	Exploring nanoparticles as CRISPR-Cas9 delivery systems ____
	2.1 Critical challenges for NP mediated CRISPR-Cas9 delivery ___________________
	2.2 Important characteristics of nanoparticle-based delivery systems __________
	2.3 Nanocarriers as optimal platforms for precise CRISPR-Cas9 delivery _______
	2.4 Unraveling the remarkable potential of peptide-based nanoparticles (PBN) as
	delivery system __________________________________________________________________________
	3.	Decoding the KRAS oncogene _______________________________________
	3.1 Discovery of KRAS ________________________________________________________________
	3.2 Navigating the biology of KRAS _________________________________________________
	3.3 KRAS mediated signaling pathway _____________________________________________
	3.4 Insights into oncogenic KRAS mutations _______________________________________
	3.5 Main KRAS-driven cancers ______________________________________________________
	3.6 Drugging the "undruggable" KRAS ______________________________________________
	3.7 Precise targeting of KRAS using CRISPR-cas Systems _________________________
	1. Innovative ADGN peptides show interesting properties for RNA delivery ____
	2. ADGN peptides effectively complex with mRNA molecules _____________________
	3.

ADGN-PBN show a monodisperse size distribution _____________________________ 4. Peptide to RNA ratio highly influence transfection efficiency __________________ 5. ADGN-PBN induced luciferase expression on cancer cells ______________________ Introduction 1.

Deciphering the CRISPR-Cas9 system for precise genome editing

Delivery mode Advantages Shortcomings DNA

	Low cost High stability	Low efficiency Delayed onset Integration risk
	mRNA
	Transient expression
	Quick onset	Poor stability
	Low off-target effects
	RNP
	Transient duration	High cost
	Quick onset	Endotoxin
	Low off-target effects	contamination

Table 2 .

 2 Characteristics of different CRISPR-Cas9 delivery modes. Adapted from (L.[START_REF] Wang | Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy[END_REF].

Table 4

 4

			gene Pancreas Lentivirus	DNA	PACO9,	(Muzumdar
				Retrovirus		PACO19	et al., 2017)
	CRISPR-	G12D	Pancreas	Lipofectamine RNP	AsPC-1,	(X. Zhao et
	Cas13a		CRC			PANC-1,	al., 2018)
			Lung			HPAF-II,
						T3M4,
						LS174T,
						SK-LU-1
	CRISPR-	G12D	Pancreas Lipofectamine DNA	Panc-1,	(Lentsch et
	Cas9					SUIT-2,	al., 2019)
						TB32047
	CRISPR-	G12D	Pancreas AAV	DNA	n/a	(W. Jiang et
	CasRX	G12C					al., 2020)
	CRISPR-	G12D	Pancreas Lentivirus	DNA	FC1199,	(Ischenko et
	Cas9					FC1242,	al., 2021)
						FC1245,
						A9312,
						A9993
	CRISPR	G12D	Pancreas Lipofectamine DNA	CFPAC-1,	(Jang et al.,
	prime	D12V				ASPC-1	2022)
	editing
	CRISPR-	G12D	CRC	Lentivirus	DNA	SW480,	(W. Lee et
	Cas9	G12V	Pancreas			SW620,	al., 2018)
						SNU407,
						AS-PC1
	CRISPR-	G12D	CRC	Lentivirus	DNA	HT29,	(W. Kim et
	Cas9	G12V				SW403,	al., 2018)
		G13D				SW480,
						SW620,
						LS513,
						LoVo
	CRISPR-	Whole gene CRC	Polymer NP	RNP	SW480	(Wan et al.,
	Cas9						2020)
	CRISPR-	Whole gene CRC	Lipofectamine RNP	HCT-116,	(J. Liu et al.,
	dCas9-		Lung			NCI-H358	2021)
	HDAC1
	CRISPR-	G12C	Lung	Lipofectamine	RNP	A549, H23 (S. M. Kim et
	Cas9	G12D		Adenovirus			al., 2018)
	CRISPR-	G12S	Lung	Adenovirus	DNA	A549,	(Gao et al.,
	Cas9 and					H2228	2020)
	dCas9-
	KRAB

. Direct targeting of KRAS in cancer via the CRISPR systems. Adapted from

[START_REF] Bender | CRISPR and KRAS: a match yet to be made[END_REF]

Table 5 .

 5 Peptide sequences

Table 7

 7

			Size Distribution by Intensity
		20
	Intensity (Percent)	5 10 15			Measure 1 Measure 2 Measure 3
		0
		1	10	100	1000
			Size (d.nm)
	Figure 39. Representative DLS intensity-based size distribution histogram of ADGN-PBN complexed with
	0.8 µg of fLuc mRNA at 2:1 peptide:RNA weigth ratio. Three different measures (in blue, red and green)
	were performed.
			Peptide:RNA ratio	2:1
			Size distribution (nm)	91.87 ± 1.5
			PDI		0.17 ± 0.02
			ζ Potential (mV)	-9.81 ± 1.5

. DLS Physical characterization of ADGN-PBN complexed with 0.8 µg of fLuc mRNA at 2:1 peptide:RNA weigth ratio.

Table 8 .

 8 DLS characterization of ADGN peptides complexed with CRISPR-Cas9 RNA components at different peptide:RNA weight ratios.

	Peptide:RNA ratio	Size distribution (nm)	PDI	ζ Potential (mV)
	0.5 : 1	109.57	0.18	-12.6
	1 : 1	106.47	0.18	-6.79
	2 : 1	139.63	0.22	7.49

Table 9 .

 9 Fluorescence intensity values of labelled ADGN-PBN formed with Cy5-labelled peptide and/or Cy3labelled sgRNA at different peptide to RNA weight ratios after excitation at 496 nm. Emission wavelength: 685 nm or 565nm.

	1 (Cy5-Cy3)	PBN 1:1 (Cy5)

Table 11 .

 11 Sequences of the sgRNA used for CRISPR-Cas9 gene editing.

  ___ 76 Table 5. Peptide sequences________________________________________________________________________________________ 81 Table 6. Conditions tested for optimal complexation of ADGN peptides with mRNA molecules. ______________ 84 Table 7. DLS Physical characterization of ADGN-PBN complexed with 0.8 µg of fLuc mRNA at 2:1 peptide:RNA weigth ratio. _______________________________________________________________________________________________________ 85 Table 8. DLS characterization of ADGN peptides complexed with CRISPR-Cas9 RNA components at different peptide:RNA weight ratios. _______________________________________________________________________________________ 89 Table 9. Fluorescence intensity values of labelled ADGN-PBN formed with Cy5-labelled peptide and/or Cy3labelled sgRNA at different peptide to RNA weight ratios after excitation at 496 nm. Emission wavelength: 685 nm or 565nm. ______________________________________________________________________________________________________ 96 Table 10. Clinical trials for disease gene editing with CRISPR-Cas9 (Carroll, 2023). _________________________ 121 Table 11. Sequences of the sgRNA used for CRISPR-Cas9 gene editing. _______________________________________ 131 Table 12. Primers used to the T7E1 DNA mismatch detection assay. __________________________________________ 136 Table 13. Sequences of the primers used for qPCR-based approach for DNA mutation detection. ___________ 136 Table 14. List of Antibodies used for WesternBlot analysis. ____________________________________________________ 138

Acknowledgments

I would like to express my sincere gratitude to all the people who have directly or indirectly contributed to the journey that has brought me to this point. Without your support, I wouldn't be where I am today.

Where the fraction cleaved is defined as: 𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑐𝑙𝑒𝑎𝑣𝑒𝑑 = 𝑐𝑙𝑒𝑎𝑣𝑒𝑑 𝑏𝑎𝑛𝑑𝑠 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑐𝑙𝑒𝑎𝑣𝑒𝑑 𝑏𝑎𝑛𝑑𝑠 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 + 𝑢𝑛𝑐𝑙𝑒𝑎𝑣𝑒𝑑 𝑝𝑎𝑟𝑒𝑛𝑡𝑎𝑙 𝑏𝑎𝑛𝑑𝑠 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 Name of the primers Sequences Luc forward 5'-CTTCCGCATAGAACTGCCTG-3' Luc reverse 5'-GTAGCCATCCATCCTTGTCA-3'

Table 12. Primers used to the T7E1 DNA mismatch detection assay.

Real time PCR amplification

In order to estimate the editing efficiency of CRISPR-Cas9 delivered by ADGN-PBN, we used a qPCR-based method that combines the sensitivity of Taq polymerase to mismatch at primer 3′ end with a qPCR technique for its power in DNA quantification. One of the 2 primers (called IN) directly binds to the expected Cas9 nuclease cutting site near its 3′ end. A primer couple (called OUT) for control amplification is introduced few base pairs away from the cutting site for normalization purpose in calculating the percentage of the edited DNA in the total genomic DNA sample. In addition, when targeting the endogenous KRAS gene, a blocking 3'-phosphate modified oligonucleotide was used to highly discriminate between the mutant and the wild type alleles. Wild type sequences are amplified with optimal PCR efficiency, in contrast to edited DNA that is partially amplified. DNA was extracted from transfected and non-transfected cells with the DNeasy blood and tissue kit from QIAGEN according to the manufacturer's instructions. Target loci from 400 ng of extracted DNA was amplified by qPCR using the ABsolute blue qPCR SYBR Green kit from ThermoScientific. qPCR was performed in a Lightcycler Roche 480 system from Roche Diagnostics with the following conditions: one cycle of 95°C for 15 min, 40 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s. The sequences of forward primers and of reverse primers are indicated in the table below:

Name of the primers Sequences

In vivo studies

Experiments were performed in the AADIGEN/LH laboratory at UCLA/Children Hospital. Female athymic NMRI nude mice 6 weeks (Charles River Laboratory) were kept under 12 h light/12 h dark cycles and were fed with a standard laboratory rodent diet in pellets form, food and water were available ad libitum. Two million of PANC1 cells or SW403 cells were suspended in Hank's Balanced Salt Solution and injected subcutaneously in the right flank. When tumors were palpable, mice were randomized into groups and treated by intravenous injection with 90μL of vehicle (physiological buffer: 0.9% sodium chloride) or ADGN-PBN (0.5 mg/kg and 1.0 mg/kg). ADGN-PBN are obtained using ADGN-ava peptide, CAS9mRNA and sgRNA at charge ratio of 5:1. Mice were treated on day 1 and 7 and were maintained in a pathogen-free environment and monitored by observation of overall health and weekly body weights to determine NPs tolerability. Tumors were measured weekly with calipers. Tumor volume was calculated by the formula V= π/6 × Length (mm) × Width2 (mm). Mice in the control/ADGN-PBN G12C groups and mice in the ADGN-PBN G12D and G12V groups were sacrificed at day 40 and 90, respectively.

Statistical analysis

Statistical analyses were performed by using Graph Pad Prism 8 software (GraphPad, San Diego, CA, USA). Data were analyzed by Mann-Whitney U test for non-parametric data one-way ANOVA with Tukey's Honestly-Significant Difference post-hoc test was used to compare three or more groups. Results are presented as mean ± SEM. P values of less than 0.05 were considered significant.

Annexes

Abstract

The clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) system has emerged as a revolutionary gene-editing tool with huge therapeutic potential to address the genetic root causing diverse diseases such as cancer. KRAS is the most frequently mutated oncogene in human cancer. Its predominant mutations at codon 12, G12D and G12V, are main drivers in common cancers, including pancreatic and colorectal cancers. KRAS mutations remain one of the most challenging cancer targets. CRISPR-Cas9 system provides an attractive opportunity to target these cancer-causing mutations. However, there are several challenging factors that must be addressed for its use in clinic. One key factor is the delivery method. In addition to the risk of nuclease degradation and rapid CRISPR-Cas9 system clearance by macrophages, the large size of Cas9, the high anionic charge density and hydrophilic nature of the RNA impose a significant barrier to their intracellular delivery and overall gene transfection efficiency.

We developed new Peptide Based Nanoparticles (PBN), a delivery system that has been identified as one of the most promising non-viral strategies for the intracelullar delivery of nucleic acids. We demonstrated that these PBN form stable complexes with Cas9 mRNA and its sgRNA and are able to protect the CRISPR-Cas9 RNA components from nuclease degradation. Using fluorescence confocal microscopy, we confirmed the intracelular delivery of the system. We first verified the delivery of a funcional CRISPR-Cas9 system by targeting the reporter gene luciferase. PBN efficiently generated the knock-out of the luciferase gene in vitro without any apparent cytotoxicity. PBN produce robust gene editing with 60% efficiency and 50% decrease of overall luciferase activity. Furthermore, PBN were able to target the predominant KRAS mutations, reaching 20% of gene editing efficiency for the G12D mutation and 60% for the G12V mutation. The phosphorylation of key downstream proteins in KRAS signaling pathway was downregulated, and cell proliferation rate was recovered after CRISPR-Cas9 editing.

We showed for the first time that peptide-based nanoparticles were able to deliver the CRISPR-Cas9 system in the form of RNA in cancer cells and to effectively target the G12D and G12V mutations of the KRAS oncogene. Our results highlight the importance of an appropriate delivery system to deliver CRISPR-Cas9 and open up promising therapeutic perspectives to target KRAS mutations involved in cancer development.