Gabriella Dias

Florencio Leite

Javier Perea

Aurélie Goyenvalle

Valérie Robin

Lucia Echevarria

Graziella Griffith

Cyriaque Beley

Pierre Olivier Buclez

Aurélie Avril

Faouzi Zarrouki

Julien Dattola

Amalia Stanzos

Jakub Melcher

Mina Petkova

Fedor Svinartchouk

Antoine De Zélicourt

Sabine De

La Porte

Philippine Lucia Sonia

Aurélie, Olivier, Benoit, Cyriaque Malika Helge Amthor

teaching and research institutions in France or abroad, or from public or private research centers.

Slavica and Emmanuel Moyse for your help in personal and science problems.

Nicolas Dauvillier for supporting me at the through the final steps of this manuscript, not an easy task! Finally, I extend my sincere thanks to my family that accepted my moving across the ocean to pursuit my dreams: My parents, my siblings, my grandmother and all my family and friends, who have accompanied me, helped, supported and encouraged throughout this journey.

Without your help this work would not have been done, THANK YOU!!

Table of Contents

LIST OF FIGURES

LIST OF TABLES TABLE 1. RECEPTORS, CO-RECEPTORS AND TISSUE TROPISMS OF THE MOSTLY

I. FROM GENE TRANSFER CONCEPT TO GENE THERAPY APPROACHES

I.1 The idea of gene therapy: a biological history of 20th century "I'm sorry: your child has a genetic disease..." These words, formulated with all the weight of fatality, have certainly resonated terribly in the minds of thousands of parents during the previous century. Until the late 1980s, the possibility of a treatment for inherited pathologies was still of the order of science fiction.

Although the notion of heredity and the concept of the gene were progressively clarified during the 20th century, the idea of correcting or complementing genetic mutations remained a major technological challenge until the 1980s. In addition to the discovery of the "laws of heredity" by Gregor Mendel in 1865 (Mendel, 1865), it was the chromosomal theory formulated by Thomas Hunt Morgan in 1910 that triggered the first revolution in understanding the transmission of genetic information from one generation to the next. The mechanics of the so-called "vertical" gene transfer was studied by the Morgan team using models of mutant flies [START_REF] Morgan | The Mechanism of Mendelian heredity[END_REF]. Very soon, the first genetic maps appeared, allowing the physical localization of the genes on the chromosomes. However, it was not until 1944 that the true support of genetic information, the deoxyribonucleic acid (DNA), was obtained, through the famous experiments of Drs Avery, MacLeod and McCarty on bacterial transformation [START_REF] Avery | STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES : INDUCTION OF TRANSFORMATION BY A DESOXYRIBONUCLEIC ACID FRACTION ISOLATED FROM PNEUMOCOCCUS TYPE III[END_REF]. It is remarkable to note that the transfer of exogenous genetic material was already practiced at this very early period of modern biology even if the mechanisms of this transfer remained to be understood and that the structure of the DNA was still not known. It took ten more years to elucidate the double helix structure of DNA after the famous work of James Watson and Francis Crick in 1953 [START_REF] Watson | Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid[END_REF]. The description of the mechanisms of genetic regulation by François Jacob and Jacques Monod in 1961 [START_REF] Jacob | Genetic regulatory mechanisms in the synthesis of proteins[END_REF] helped to consolidate researchers' understanding on the nature of genetic material, its role and especially its molecular mechanism (transcription and translation processes).

The late 1960s saw the emergence of the concept of gene transfer in higher mammalian cells by "transforming" viruses such as SV40 (Simian virus 40) [START_REF] Sambrook | The integrated state of viral DNA in SV40-transformed cells[END_REF]. Their ability to integrate their viral genome into the targeted cell genome has raised the interest of researchers to use these biological "vehicles" to transport genes of interest and to express them in the cells or tissues of their choice. However, the lack of robust tools and techniques capable of providing artificial genetic constructions constituted a real obstacle to the development of this gene transfer strategy. The discovery of restriction enzymes in bacteria by Werner [START_REF] Arber | Host-controlled modification of bacteriophage[END_REF][START_REF] Arber | Host-controlled modification of bacteriophage[END_REF], and especially their use for DNA mapping by [START_REF] Danna | Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae[END_REF][START_REF] Danna | Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae[END_REF][START_REF] Danna | Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae[END_REF] have truly opened the way to recombinant DNA technology and genetic engineering. They allowed the first molecular cloning the following year [START_REF] Jackson | Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli[END_REF]. It is the enthusiasm aroused by these technological developments that naturally led researchers to propose therapeutic strategies for genetic diseases based on gene transfer. A famous article published in 1972 by Theodore Friedmann and Richard Roblin in Science set the tone with an explicit interrogative title: "Gene Therapy for Human Genetic Disease?" [START_REF] Friedmann | Gene therapy for human genetic disease?[END_REF].

The ethical and societal questions raised by these approaches and the new therapeutic perspectives immediately led the researchers to establish a moratorium to discuss all these aspects during a conference that was renowned for the novelty of its thematic: The Asilomar Conference of 1975. In spite of the small concrete spinoffs, the conference had the merit of alerting the safety aspects generated by the emergence of new genetically modified organisms (GMOs). The urgent measures that the states will have to put in place to minimize the risks of dissemination of these products, particularly in the human population, had been discussed.

I.2 The natural gene transfers: the "horizontal" point of view

I.2.1 The "horizontal" gene transfer: generalities

Vertical gene transfer, briefly referred above, is defined as the transmission of genetic material from an organism or cell to a descendant. It is achieved through reproduction, whether it is sexual or asexual. It differs from horizontal gene transfer (HGT), which is defined as a process in which an organism incorporates genetic material from another organism without being descended from it. It is through the study of certain "natural" HGTs that the idea of gene therapy has gradually entered the minds of scientists. Some examples are given in the following section.

-The transduction: the genetic material of a bacterium is transported in another bacterium through a virus (bacteriophage). This pathway of genetic material transfer is particularly important in the studies that will be presented later and will be the subject of a specific development when we address viral vectors of gene transfer.

b. HGT in eukaryotes

The HGT in eukaryotes has long been considered a very rare phenomenon.

However, with the emergence of high-throughput sequencing techniques, numerous evidence of HGT has begun to be reported in eukaryotes, even putting into difficulty classical phylogenetic patterns [START_REF] Soucy | Horizontal gene transfer: building the web of life[END_REF]. The following HGTs in eukaryotes have been described [START_REF] Boto | Horizontal gene transfer in the acquisition of novel traits by metazoans[END_REF]:

-between bacteria and sponges, corals, fungi, yeasts, nematodes, crustaceans, plants or vertebrates -between insects and nematodes -between fungi and insects or crustaceans A particular case of HGT in animals is that attributed to the infection of different species by retroviruses. This family of viruses, as we shall see, has the unique property of integrating its viral genome into the cell genome and thus contributed to significant genetic modifications during the evolution of the species.

Although the discovery of the importance of "natural" gene transfer in eukaryotes is relatively recent, it nevertheless contributes to the understanding and especially to the development of the "artificial" transfer of genes that will be discussed in the next section.

I.3 Gene therapy: clinical application of the gene transfer strategies

Some genetic diseases are being successfully treated with metabolic manipulation and protein compensation, however, for most part no treatment is available today. The development of genetic medicines will represent a major advance in this field. Gene therapy is the replacement of a gene or the inactivation of disease causing genes and may be the hope for the treatment of monogenic diseases, a few acquired diseases [START_REF] Stayte | Advances in non-dopaminergic treatments for Parkinson's disease[END_REF], infections [START_REF] Balazs | Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission[END_REF][START_REF] Joseph | Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity[END_REF][START_REF] Tebas | Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV[END_REF], and generation of vaccines for infectious diseases [START_REF] Collins | Gene therapy meets vaccine development[END_REF]. According to OMIM (The Online Mendelian Inheritance in Man), mutations in 3311 genes are responsible for 4895 known single-gene disorder phenotypes (OMIM GENE updated on January 16 th 2017, accessed on January 17 th 2017).

The number of gene therapy clinical trials is increasing each year according to the Wiley Database Online provided by the Journal of Gene Medicine: until august 2016 approximately 2500 clinical trials were approved. The first gene therapy experimentation in humans was attempt in 1980 and was marked by violation of guidelines for not having permission [START_REF] Beutler | the cline affair[END_REF]. The absence of positive outcomes together with the death of a patient by adverse reactions resulted in the sanction of the main researcher in the project [START_REF] Sun | Cline loses two NIH grants[END_REF]. From the beginning the use of gene therapy is accompanied by ethical debates [START_REF] Murray | Human gene therapy, the public, and public policy[END_REF]. The first official clinical trial received approval in 1988 for patients with advanced melanoma [START_REF] Rosenberg | Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction[END_REF]. This trial using retroviral vectors to transduce autologous tumor infiltrating lymphocytes (TILs) reinjected into patients, was concluded without incidents and considered safe [START_REF] Wirth | History of gene therapy[END_REF]. The first in vivo gene delivery was performed in 1995 [START_REF] Puumalainen | Beta-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses[END_REF] to transfer the beta-galactosidase gene to human malignant glioma mediated by retroviruses and adenoviruses. No adverse effects were observed, however the treatment had no efficacy. The absence of results did not discourage researches, especially because so far, the use of viral vectors to transfer gene did not show adverse reactions. It was not until the end of 90's that attention would turn to the major safety concerns around this technology. One clinical trial in the University of Pennsylvania (United States) would cause the death of 18-year-old Jesse Gelsinger, a patient with partial ornithine transcarbamylase (OTC) deficiency. Patients with this disease usually die before the age of 5. In 1999, he was enlisted in a clinical trial in which he received corrective OTC mediated by a recombinant adenoviral vector directly into the blood stream. Adverse severe immune reactions resulted in death of this patient four days after the injection [START_REF] Sibbald | Death but one unintended consequence of gene-therapy trial[END_REF]. This incident led the United Stated Food and Drug Administration (US FDA) and the National Institute of Health (NIH) to create two programs in order to enhance patient safety: Gene Therapy Clinical Trial Monitoring Plan and the Gene Transfer Safety Symposia.

In parallel, in Europe, two teams attempted to treat X-linked SCID in two groups of patients (10 in Paris and 10 in London) by the ex vivo correction of CD34 + patient cells treated with a cDNA vectorized by defective murine leukemia virus (MLV) vector and gibbon-ape leukemia virus (GALV)-pseudotyped retrovirus respectively [START_REF] Cavazzana-Calvo | Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease[END_REF][START_REF] Hacein-Bey-Abina | Efficacy of gene therapy for Xlinked severe combined immunodeficiency[END_REF].

Altogether, the trials were successful in treating 17/20 patients enrolled, that displayed sustained presence of transduced lymphocytes after 11 years of treatment. Five patients, however, developed T-cell leukemia 2 after five years of treatment due to provirus integration in a pro-oncogenic locus. Following chemotherapy, 4 out of the 5 patients showed remission, but 1 patient died from refractory leukemia [START_REF] Fischer | Gene therapy for primary adaptive immune deficiencies[END_REF].

The first marketing approval for a gene therapy product was given by the Chinese State Food and Drug Administration in 2003, named Gendicin ® . This product developed by SiBiono Gene Tech Co. carries a human p53 cDNA vectorized by Adenovirus to treat head cell carcinomas [START_REF] Peng | Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers[END_REF]. Even though no phase III clinical trial data was presented before approval [START_REF] Xin | Chinese gene therapy. Gendicine's efficacy: hard to translate[END_REF], this product is still on the market today. Still in China, another marketing approval for the treatment of late-stage refractory nasopharyngeal cancer was approved in 2005.

At this time another product, called Oncorine ® composed with a conditionally replicative adenovirus, specific to replicate in p53 deficient cells, was used to provoke an efficient apoptosis of the targeted cells. [START_REF] Liang | Clinical development of oncolytic viruses in China[END_REF].

In the European Union, the first application for marketing authorization for genetic medicine was made in 2009 for treatment of malignant brain tumors. This product developed by Ark Therapeutics Group plc is called Cerepro Ò . It consists of adenoviral vectors carrying a gene from the Herpes simplex virus thymidine kinase (HSV-tk) conceived to be injected directly into the tumor [START_REF] Wirth | History of gene therapy[END_REF].

However, application for marketing authorization for this product was withdrawn in 2010 by Ark Therapeutics Group plc for lack of clinical benefits.

The first marketing approval in the European Union for the use of a recombinant adeno-associated virus, named Glybera ® (alipogene tiparvovec) (Ylä-Herttuala, 2012), was given in 2012, to restore the LPL enzyme activity in a familial lipoprotein lipase deficiency [START_REF] Gaudet | Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial[END_REF].

To resume, today there are seven gene therapy products mediated by viral vectors: Gendicine ® was approved in China in 2003 and developed by SiBiono Gene Tech Co. [START_REF] Peng | Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers[END_REF]; Oncorine ® commercialized by Shanghai Sunway Biotech Co.

was approved in China in 2005 [START_REF] Liang | Clinical development of oncolytic viruses in China[END_REF]; Rexin-G ® produced by Epeius Biotechnologies Corporation was approved in the US in 2007 [START_REF] Gordon | Rexin-G, a targeted genetic medicine for cancer[END_REF]; Glybera ® , approved in the EU in 2012 was developed by Amsterdam Molecular Therapeutics (Ylä-Herttuala, 2012); Imlygic ® approved in the US in 2015 commercialized by BioVex Inc. [START_REF] Pol | First oncolytic virus approved for melanoma immunotherapy[END_REF], and the last two approved in the EU in 2016 Strimvelis ® produced by GlaxoSmithKline and Zalmoxis ® produced by MolMed S.p.A [START_REF] Farkas | Advanced Therapy Medicinal Products for Rare Diseases: State of Play of Incentives Supporting Development in Europe[END_REF]. After almost 30 years since the first human gene therapy clinical trial, efforts are starting to pay up as the first gene therapy treatments mediated by viral vectors are in the market.

I.4 The artificial gene transfers: the science of vectorology

I.4.1 The "transgene" concept and the molecular cloning

Transgenes are engineered cDNAs which can be inserted into expression cassettes. In addition to the transgene, the cassette must contain a promoter, and optional features such as polyadenylation sequences, and regulation sequences in order to obtain the expected gene expression [START_REF] Haruyama | Overview: Engineering Transgenic Constructs and Mice[END_REF]. Transgenes can have a therapeutical function, reporter function or play a regulatory role in the expression of other genes. These can be used in basic research, for the identification and characterization of gene function, or in gene therapy, to revert disease phenotype.

The choice of promoter is particularly important for the spatial (tissue-specific) and temporal (e.g. developmental stage-specific) regulation of gene expression.

The engineering of these transgenes and expression cassettes, termed molecular cloning, is due to the discovery of restriction enzymes in 1960's [START_REF] Arber | Host-controlled modification of bacteriophage[END_REF]. These enzymes are endonucleases which allow sequence specific cleavage of DNA, thus the first step in the advance of the recombinant DNA technology [START_REF] Loenen | Highlights of the DNA cutters: a short history of the restriction enzymes[END_REF]. This technology was crucial for the development of the molecular tools required for gene transfer.

I.4.2 Gene transfer vectors: generalities

One of the main challenges today is the delivery of the molecular tools suitable for gene therapy of targeted tissues. Efficiency of in vivo gene therapy is determined by the method or vector used to deliver the transgene, its mode of administration, and the nature of the target cell [START_REF] Nishikawa | Nonviral Approaches Satisfying Various Requirements for Effective in Vivo Gene Therapy[END_REF]. Gene transfer vectors are tools used to facilitate the transport of a transgene to specific cells, with the challenge of crossing cell membrane and deliver the therapeutic DNA into the nucleus where gene translation occurs. Gene transfer vectors (non-viral or viral vectors) must be suitable to target disease/target cell and offer efficient and longterm gene expression. The ideal strategy must be non-toxic, well characterized and which has already showed proof of principle in an experimental animal model of the disease [START_REF] Hauswirth | Ocular gene therapy: quo vadis?[END_REF]. The first in vivo gene transfer using naked DNA, published in 1990, achieved to deliver transgenes into mouse skeletal muscle [START_REF] Wolff | Direct gene transfer into mouse muscle in vivo[END_REF]. Each strategy displays advantages and disadvantages, and the types of vectors will be further explained in the following chapter.

I.4.3 Non-viral vectors: types and mode of action a. Delivery methods of non-viral tools

The non-viral delivery systems can be divided into two categories: physical and chemical methods. Physical delivery methods employ a physical force to increase permeability of the cell membrane. Among the advantages of these types of delivery methods are the safety, the efficiency to transfer the transgene to cells in vitro and in vivo and the possibility to transfect dividing and non-dividing cells [START_REF] Kafri | Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors[END_REF]. On the other hand, chemical methods employ carriers such as cationic lipids or polymersomes to help the entry to the target cell. These techniques are safer than viral vectors [START_REF] Gao | Nonviral gene delivery: What we know and what is next[END_REF], however, display low transfection rate in vivo and in non-dividing cells and some compounds provoke acute toxicity that can be detrimental for the injected organisms [START_REF] Alsaggar | Physical methods for gene transfer[END_REF].

b. Delivery of naked DNA

The application of gene transfer using naked DNA can be attractive strategy when compared to viral-mediated methods, since some specific concerns such as high cost of recombinant viral-vector production and immunogenicity of viral proteins can be avoided. However, the efficiency by which naked DNA can bypass cellular membranes in vivo is usually poor [START_REF] Gao | Nonviral gene delivery: What we know and what is next[END_REF]. This is especially problematic for diseases in which a high number of cells must be transfected or when a large gene has to be introduced; in these cases, this technique shows usually little clinical utility [START_REF] Nowak | Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment[END_REF]. Intravenous injection of large genes using naked DNA is a challenge and mechanisms leading to DNA uptake following the pH drops and calcium phosphate dissolves, freeing the DNA. The dissolved calcium phosphates destroys the endosome and DNA is released into the cytosol [START_REF] Lee | Nano-sized calcium phosphate (CaP) carriers for non-viral gene deilvery[END_REF]. The simplicity is one of the main advantage, however, its use is limited to in vitro DNA transfer.

d. Delivery of DNA by cationic lipids, liposomes and lipoplexes

Liposomes are sphere-shaped vesicles consisting of one or more phospholipid bilayers that can vary from very small size (0,025µm) to large (2,5µm) particles [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. Formed by a hydrophilic interior and hydrophobic membrane, this technology is able to transport transgenes protected from the physiological environment. Liposomes received the first marketing approval for clinical in 1995 for the polyethylene glycolated liposomal formulation Doxil ® and since then several formulations are currently evaluated for their use in future clinical trials [START_REF] Bulbake | Liposomal Formulations in Clinical Use: An Updated Review[END_REF].

Several mechanisms of liposome-cell interaction can take place for the transfer to occur: (1) the fusion of the liposome with the plasma membrane (Figure 3); [START_REF]Pre-lysis: Add 180µL Buffer T1 and 25µL Proteinase K solution. Vortex to mix. Be sure that the samples are completely covered with lysis solution. Incubate at 56°C[END_REF] endocytosis-mediated uptake; and (3) receptor-mediated liposome uptake (Düzgüneş[START_REF] Duchenne | De la paralysie musculaire pseudo-hypertrophique, ou paralysie myo-sclérosique / par le Dr Duchenne (de Boulogne). Düzgüne&scedil[END_REF]. The lipid composition is the main parameter that can influence the cellular uptake of these carriers. In the context of gene transfer, cationic liposomes are the most employed. The use of cationic liposomes is based on the interaction of a lipid-DNA complexes with the target cell membrane. Due to its lipophilic nature, these formulations can entry the cell releasing the DNA, RNA or drug molecules inside the cell (Figure 3), or this complex will undergo endosomal uptake and be transferred to the nucleus where the therapeutic DNA can interact with the genetic material.

Liposomal based strategies present advantages and disadvantages. Among its advantages we can name the increase stability following encapsulation, low toxicity, non-immunogenic, biocompatibility and increased efficacy when compared to injection of naked drugs or naked DNA. Among the disadvantages there are: low solubility, short half-life, possible oxidation of phospholipids in the composition, possible fusion with drugs/molecules encapsulated, high cost of production [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF].

e. Delivery of DNA by polymersomes

Polymersomes are engineered biocompatible nanoparticles composed of amphiphilic block copolymers that can mimic cell membranes [START_REF] Nance | Nanotherapy for Duchenne muscular dystrophy[END_REF].

c. Delivery of transgenes by Retrovirus/Lentivirus vectors

From the Retroviridae family, Gammaretrovirus (MLV -Murine Leukemia Viruses) and Lentivirus are single stranded RNA viruses. These viruses present surface glycoproteins that recognize receptors on the cell membrane, promoting their entry into the cell cytoplasm. Once inside the cell, the enzyme retrotranscriptase produces double stranded DNA from the single stranded RNA molecule, and is integrated to the host genome by the action of the integrase enzyme [START_REF] Naldini | In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[END_REF]. Retroviral vectors were the most preferred gene transfer systems in clinical gene therapy until the incident of a human trial for the X-linked severe combined immunodeficiency (SCID) in which four out of nine successfully treated patients later developed leukemia [START_REF] Cavazzana-Calvo | Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease[END_REF]. Retrovirus present two major safety issues: (1) The potential risk of insertional oncogenesis and ( 2) the possibility of generating replication competent retroviruses (RCR) [START_REF] Hacein-Bey-Abina | LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1[END_REF]. Even though new generations of retroviral vectors are designed to reduce the risk of RCR, efforts have not yet eliminated this problem [START_REF] Anson | The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery[END_REF]. These high safety issues can be a major problem when considering in vivo treatments.

Lentivirus' capacity to produce Replication Competent Lentiviruses (RCL) is very low with current viral vector systems [START_REF] Cornetta | Replication-competent lentivirus analysis of clinical grade vector products[END_REF], but they present the insertional risk. Several generations of lentiviral vectors have been constructed to improve their safety and efficiency [START_REF] Kumar | Optimization of lentiviral vectors generation for biomedical and clinical research purposes: contemporary trends in technology development and applications[END_REF]. This characteristic may, however, be beneficial when specific and stable long-term expression of the transgene is needed for achieving therapeutic efficacy. This is particularly true for dividing tissues as for the hematopoietic system. New generation of integrase-defective LV (IDLVs) vectors offers an interesting option for a better use of these vectors in vivo [START_REF] Joglekar | Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus[END_REF]. In LV vectors, the packaging capacity is around 8kb [START_REF] Kumar | Optimization of lentiviral vectors generation for biomedical and clinical research purposes: contemporary trends in technology development and applications[END_REF]. Their structure is schematized in figure 5.

INTRODUCTION

II. Recombinant Adeno-Associated Virus: an attractive viral vector for gene therapy

II.1 Wild type Adeno-associated viruses

AAVs were first observed in 1960's in mixtures with Adenovirus, hence the name [START_REF] Atchison | Adenovirus-Associated Defective Virus Particles[END_REF][START_REF] Hoggan | Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics[END_REF]. At first, they were thought to be either contaminant particles or adenovirus sub particles [START_REF] Atchison | Adenovirus-Associated Defective Virus Particles[END_REF]. . Because AAVs are not able to replicate by themselves, these virus depend on helper functions offered by other viruses, such as Adenovirus, Herpesvirus or Papillomavirus, which made them be classified in the genus Dependovirus [START_REF] Carter | Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective[END_REF].

II.1.1 Structure, genome, serotypes and tropism

AAVs are non-enveloped virus composed of an icosahedral capsid of approximately 26nm in diameter. The AAV genome is a well-characterized linear single stranded DNA of 4,7 kb, flanked by two inverted terminal repeats (ITRs) [START_REF] Srivastava | Nucleotide sequence and organization of the adeno-associated virus 2 genome[END_REF]. The AAV genome carries the rep and cap genes, that codes for viral enzymes and the capsid structure proteins, respectively. The capsid is composed by three proteins, called viral proteins (VPs) in a well-defined ratio: VP1, VP2 and VP3 (1:1:10) (Figure 8). These proteins are the main determinant for AAV serotype and tissue tropism.

Today there are twelve human serotypes of AAV identified (AAV serotype 1 to 12) and more than 100 serotypes from nonhuman primates [START_REF] Daya | Gene therapy using adeno-associated virus vectors[END_REF][START_REF] Gao | Exploiting natural diversity of AAV for the design of vectors with novel properties[END_REF]. Depending on the serotype, different mechanisms, receptors and coreceptors are implicated in AAV entry on the cell. The main serotypes used in research are listed on the table below (Table 1). During the lysogenic phase, AAV is latent and no replication occurs. During this phase AAV integrates the host genome in a well identified locus on the chromosome 19 (q13.4) named AAVS1 [START_REF] Kay | Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector[END_REF].

II.1.3 Immunity and seroprevalence

One of the downside of viral vectors is the immune response elicited following injections. Immune response against viruses can be either cytotoxic (destruction of cells expressing the transgenes) or humoral (production of antibodies against the viral capsid/envelop/surface proteins) [START_REF] Daya | Gene therapy using adeno-associated virus vectors[END_REF][START_REF] Mechnikov | Immunity In Infective Diseases[END_REF]. In the case of AAV the immune response is mainly humoral, and it represents one of the highest seroprevalence in the human population [START_REF] Rapti | Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models[END_REF]. The mean seroprevalence according to several publications is between 20-40% [START_REF] Halbert | Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors[END_REF][START_REF] Rapti | Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models[END_REF], however it can reach up to 67% in some populations [START_REF] Ferla | Prevalence of Anti?Adeno-Associated Virus Serotype 8 Neutralizing Antibodies and Arylsulfatase B Cross-Reactive Immunologic Material in Mucopolysaccharidosis VI Patient Candidates for a Gene Therapy Trial[END_REF]. The most common strategy to bypass the immune response is the previous transient treatment with immunosuppressant. Results from studies on non-human primates revealed that immunosuppression treatment may lead to impaired transduction levels by AAV vectors [START_REF] Jiang | Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy[END_REF][START_REF] Mingozzi | Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver[END_REF][START_REF] Unzu | Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in nonhuman primates[END_REF]. In addition, a strong downside for this strategy is the susceptibility of patient for infections. Another strategy has been tested in animals to reduce the destruction of AAV vectors: the use of decoy capsids (empty AAV capsids) at the same time to compete for the circulating antibodies [START_REF] Mingozzi | Overcoming preexisting humoral immunity to AAV using capsid decoys[END_REF]. This strategy, however, has not yet been deemed safe in humans so far. This represents today one of the major challenges for the use of AAV vectors in gene therapy.

II.2 Recombinant AAVs

The interest in rAAV vectors for research and clinical purposes in the treatment of genetic diseases have led to the rapid evolution of methods for AAV production in the last two decades [START_REF] Ayuso | Production, Purification and Characterization of Adeno-Associated Vectors[END_REF]. As already described, these viruses present several advantageous characteristics for the use as vectors. To name a few of these characteristics: (1) these are non-pathogenic viruses; (2) they are capable of infecting both dividing and non-dividing cells; (3) the variety of different serotypes can increase tissue specificity; (4) they induce lower immune reactions than other viral vectors. Among the methods, tri-transfection of human embryonic kidney 293 (HEK293) cells remains the most popular for research scale [START_REF] Grimm | The low abundance of U7 snRNA is partly determined by its Sm binding site[END_REF][START_REF] Salvetti | Factors Influencing Recombinant Adeno-Associated Virus Production[END_REF][START_REF] Xiao | Production of high-titer recombinant adenoassociated virus vectors in the absence of helper adenovirus[END_REF].

II.2.1 Research scale processes: the tri-transfection method

The tri-transfection method is based on the co-transfection HEK293 cells with three plasmids (Figure 9) [START_REF] Grieger | Production and characterization of adenoassociated viral vectors[END_REF]. The plasmid carrying the transgene must be flanked by the viral inverted terminal repeats (ITR) sequences in order to be packaged in the rAAV. The second plasmid encodes the rep and cap genes to express the viral enzymes and the capsid structure proteins respectively. Finally, the adenoviral helper plasmid will express the genome replication helper proteins.

This co-transfection is performed 3 days after seeding a multilayer cell plate. The upstream production process lasts usually 72h, after which the culture is ended and vectors are recovered from the cell pellet after centrifugation [START_REF] Grieger | Production and characterization of adenoassociated viral vectors[END_REF].

In order to release the vectors from the cells, chemical and physical treatments are required and production is then clarified by several steps of filtration (Figure 9).

Once these steps are done, the downstream process starts. Because of the presence of rAAV released in the supernatant, few teams opted for harvesting these vectors directly from the supernatant, rather than from the cell pellet [START_REF] Doria | AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently[END_REF][START_REF] Lock | Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale[END_REF][START_REF] Vandenberghe | Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing[END_REF].

The downstream processes can be performed by different methodologies, but density gradient ultracentrifugation remains the most used method for research grade rAAV purification. At the end of ultracentrifugation, one specific fraction, containing the rAAVs, is recovered. Among other methods of clarification there are the more classical ultracentrifugation on cesium chloride (CsCl2) [START_REF] Hermens | Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System[END_REF][START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF], and different chromatographic methods, using either ion-exchange [START_REF] Okada | Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes[END_REF][START_REF] Qu | Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography[END_REF][START_REF] Urabe | Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression[END_REF] or affinity-based approaches [START_REF] Anderson | A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction[END_REF][START_REF] Auricchio | Isolation of Highly Infectious and Pure Adeno-Associated Virus Type 2 Vectors with a Single-Step Gravity-Flow Column[END_REF][START_REF] Koerber | Engineering Adeno-Associated Virus for One-Step Purification via Immobilized Metal Affinity Chromatography[END_REF][START_REF] Smith | A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells[END_REF][START_REF] Cecchini | Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02-to 200-Liter Cultures[END_REF]. However, unlike ultracentrifugation, chromatographic techniques are more serotype specific and need more technical adjustments to separate the full particles from the empty ones. In addition, these techniques are more expensive to implement than the density gradient ultracentrifugation. This tri-transfection method of rAAV vector production is most convenient for in vitro approaches and for small animals testing due to the low final vector quantity recovered. To circumvent the problem of scalability of vector production, another process has been performed through the use of baculovirus/insect cell system. This will be presented in the next section.

large quantities of viruses. However, cryopreservation of rBACs causes a significant decrease in baculovirus titres at the time of thawing and use. One way to efficiently cryopreserve the rBACs is to infect insect cells and cryopreserve them early after infection (before the beginning of the lytic cycle). This system allows to generate insect cells containing rBACs [START_REF] Wasilko | The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus[END_REF]. These cells, called BIIC (Baculovirus-Infected Insect Cells), constitute the real raw material for the production of rAAV. The BIICs are then used directly in co-culture with wild-type insect cells, as schematized in figure 11. The system of production is based on the ability of rBACs to carry out an infectious cycle (identical to wild-type viruses) in insect cells before being released into the extracellular medium (by budding). The new rBACs infect new cells by a chain reaction which terminates when all cells of the culture have been infected and lysed. Among the few insect lines available, Sf9

(derived from Spodoptera frugiperda butterfly cells) are the most widely used.

As shown schematically in figure 11, the currently most used rAAV production systems operate through a co-culture of two BIICs: a BIIC-Rep / Cap releasing rBAC-Rep / Cap coding for the rep and cap genes of AAV, and a rBAC-ITR-Transgene-ITR releasing BIIC-ITR-Transgene-ITR coding for the AAV genome of interest flanked by ITRs. It is interesting to note that the baculovirus / insect cell system does not need helper genes for the production of rAAVs. The latter are provided by some unidentified genes of the baculoviral genome. The high productivity of the BIIC system is due to the strength of the promoters that control the expression of the rep and cap genes. Indeed, viral promoters P10 (normally coding for fibrous microtubule binding proteins) and Polh (coding for polyhedrin) are both non-essential for the infectious cycle of rBAC; however, they play an important role in the dissemination of wild-type baculovirus in the natural environment.

The co-infection of a Sf9 cell by the two types of rBAC described above leads to the synthesis of rAAV. Another advantage of this production system is the ability to rapidly scale up production due to the culture conditions of Sf9 cells which proliferate in suspension. Moreover, the specific culture medium, which will be described in detail in the results section, is serum-free, facilitating the purification conditions. These culture conditions allow the possibility to switch from a production system for research scale, carried out in vials, to bioreactor production system. The implementation of such a production system has thus made it possible to surpass a technological limitation in the perspective of industrial productions of gene therapy rAAV products. characterized by progressive muscle-wasting. This is the most prevalent among the rare diseases, affecting 1 in 3,500 live male births [START_REF] Zhou | Targeting fibrosis in Duchenne muscular dystrophy[END_REF]. It was first described as pseudo-hypertrophic muscular paralysis by Duchenne de Boulogne [START_REF] Duchenne | De la paralysie musculaire pseudo-hypertrophique, ou paralysie myo-sclérosique / par le Dr Duchenne (de Boulogne). Düzgüne&scedil[END_REF] and Gowers [START_REF] Gowers | A Study of the so-called Tendon-Reflex Phemomena[END_REF]. First symptoms often appear between ages of 2-5 years, and are characterized by waddling gait and difficult to get up, followed by the appearance of Gower's sign (caused by weakness in lower limb muscles, patients must use the arms to pull their body to stand), muscle atrophy, muscle weakness and finally kyphoscoliosis (Figure 12) [START_REF] Dubowitz | Muscle disorders in childhood[END_REF].

DMD patients takes longer to acquire simple motor skills, such as walking and running, and are usually wheelchair-bound between the ages of 10 and 15 years.

The average life expectancy today is 20 to 30 years and cardiac failure associated with respiratory complications are the main causes of death among DMD patients [START_REF] Birnkrant | The respiratory management of patients with duchenne muscular dystrophy: a DMD care considerations working group specialty article[END_REF].

For instance, in the absence of dystrophin, the linkage between the cytoskeleton and the extracellular matrix is broken, and the mechanical force exerted during muscle contraction generates micro lesions of the sarcolemma (Yoshida and Ozawa, 1990). The skeletal muscle fiber is weakened by the absence of these proteins and is no longer able to perform its role. In addition, the increased membrane permeability facilitates the increase in Ca 2+ levels (Ervasti et al., 1990).

Indeed, the dysregulation of calcium homeostasis is an early event in pathophysiology and relatively common in muscular dystrophies. This increase, both during activity and rest, triggers the activation of several signalling pathways, including calcium-dependent calpain, which degrades proteins and contributes to mitochondrial mediated cell death (Ervasti et al., 1990). Other players involved in the DMD pathology are proteolysis, oxidative damage, inflammation, apoptosis, regeneration, and fibrosis [START_REF] Blake | Function and genetics of dystrophin and dystrophin-related proteins in muscle[END_REF].

b. The dystrophin gene

This disease is caused by mutations in the largest human gene that encodes a 427kDa cytoskeletal protein called dystrophin. Located on the short arm of the X chromosome, the dystrophin gene measures approximately 2.3 Mbp and is composed of 79 exons [START_REF] Monaco | Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene[END_REF]) (Figure 13).

This gene codes for a 14kb mRNA transcript with seven promoters yielding seven different dystrophin isoforms [START_REF] Blake | Function and genetics of dystrophin and dystrophin-related proteins in muscle[END_REF]. Three promoters codes for the full-length dystrophin (427 kDa) in different cell types such as striated muscle fibers, the brain and Purkinje cells, and four promoters codes for short isoforms starting at different exons and yielding the retinal (260 kDa), brain (140 kDa), Schwann cells (116k Da) and general tissues (71 kDa) isoforms [START_REF] Blake | Function and genetics of dystrophin and dystrophin-related proteins in muscle[END_REF] (Figure 13).

Out-of-frame mutations in the dystrophin gene leads to mRNA or protein instability, absence or drastic reduction in dystrophin levels and consequently to the DMD pathology. In-frame mutations lead to the expression of a truncated but functional protein. Carriers of those in-frame mutations, named Becker Muscular Dystrophy (BMD), display a milder dystrophy with later onset and longer life expectancy.

c. The dystrophin protein and dystrophin-associated protein complex (DAPC)

The dystrophin protein promotes a link between the cellular cytoskeleton and the extracellular matrix, therefore essential for the maintenance of the integrity of cardiac and skeletal muscle cells [START_REF] Lovering | The muscular dystrophies: from genes to therapies[END_REF] (Figure 14).

Dystrophin is a complex protein member of the β-spectrin/α-actinin protein family, composed by utrophin, dystrophin-related protein 2 (DRP2), and α-and βdystrobrevin. This family is characterized by the presence of an actin-binding domain located at the NH2 terminal, a large ROD domain, composed of 24 spectrinlike repeats, and thought to act as a flexible spacer between the N-terminal actin binding domain and the C-terminal domains [START_REF] Blake | The neurobiology of duchenne muscular dystrophy: learning lessons from muscle?[END_REF], and Cterminal domain composed by a WW domain, a Cysteine-rich binding domain and a ZZ domain. The WW domain is a protein-binding module implicated in the interaction between dystrophin and b-dystroglycans [START_REF] Ilsley | The WW domain: linking cell signalling to the membrane cytoskeleton[END_REF]. The cysteine domain is composed by two EF hand motifs, capable of binding intracellular Ca 2+ . Finally, the ZZ domain is similar to many types of zinc finger and is found both in nuclear and cytoplasmic proteins and binds to calmodulin in a Ca 2+ -dependent manner [START_REF] Anderson | Ca2+-calmodulin binds to the carboxylterminal domain of dystrophin[END_REF]. The domains present in the Cterminal part are characteristic of the dystrophin family of related proteins.

As already described, dystrophin is found in the plasma membrane (sarcolemma) of muscle fibers associated with several proteins, forming a complex that links the cell cytoskeleton to the extracellular matrix. This complex is called dystrophin-associated protein complex (DAPC) and can be divided into three subcomplexes: the dystroglycan sub-complex (α and β-dystroglycan), the sarcoglycan sub-complex and the cytoplasmic sub-complex composed by dystrophin, dystrobrevins and the syntrophin protein family [START_REF] Yoshida | Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n-octyl beta-D-glucoside[END_REF] (Figure 14).

There is evidence that the DAPC plays a role in protecting the sarcolemma from mechanical damage and in maintaining the calcium homeostasis. Since the dystrophin is the main protein holding the complex together, its absence leads to the instability and disassembly of the DAPC, increased membrane permeability, disruption of calcium homeostasis and consequently progressive muscle loss.

Dystrophin loss is also accompanied by reduced amounts of other DAPC proteins [START_REF] Ohlendieck | Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle[END_REF]. Disruption of other DAPC proteins is also associated to other types of dystrophies, for instance, the absence of sarcoglycans in Limb-girdle muscular dystrophy (LGMD) [START_REF] Hack | Sarcoglycans in muscular dystrophy[END_REF].

III.1.2 Genetic and differences between Becker muscular dystrophy and Duchenne muscular dystrophy

The well-known TREAT-NMD DMD global database presents data from 7.149 DMD patients. About 80% of these patients present large mutations, and 70% of those are caused by dystrophin gene deletions, leading to the loss of one or more exons [START_REF] Aartsma-Rus | Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule[END_REF]. Because of dystrophin's large size, the different mutations may result in different phenotypes. In order to produce a functional protein, the in-frame transcript must contain both the N-and C-terminal domains of dystrophin to assure the protein-protein interactions of the DAPC and provide stability to muscles fibers by linking the cytoskeleton to the sarcolemmal membrane. Mutations in the N-terminal domain are implicated in higher incidence of cognitive impairment in DMD patients [START_REF] Bushby | Genetic and clinical correlations of Xp21 muscular dystrophy[END_REF][START_REF] Lenk | Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in DMD patients[END_REF]. The most common deletions are located between exons 44-52, located in the ROD domain of the dystrophin. In frame mutations in the ROD domain however, will produce a shorter but functional protein, resulting in a milder form of dystrophy:

Becker Muscular Dystrophy (BMD).

The idea that exon-skipping could be a useful tool in treating DMD patients came from lessons learned by analyzing the BMD phenotype. Indeed, the in-frame mutations of the BMD patients provoke a milder phenotype. The conversion of an out-of-frame DMD into an in-frame BMD mutation by exon-skipping is currently one of the most researched and promising therapeutic approach for DMD (Figure 15) and will be presented in following chapters. functional or unstable protein that will be immediately destroyed (left panel). An antisense oligonucleotide that will mask and promote the deletion of exon 23 will give rise to the production of an internally truncated protein, with a small deletion in the rod domain, but remains functional.

III.1.3 Animal models

The dystrophin gene is highly conserved among vertebrates and is also found in Caenorhabditis elegans and Drosophila melanogaster [START_REF] Collins | Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies[END_REF].

Natural mutations in the dystrophin gene were reported in a few vertebrates, including mice (mdx mouse model) [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF] and dogs (GRMD) [START_REF] Kornegay | The golden retriever model of Duchenne muscular dystrophy[END_REF][START_REF] Kornegay | Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies[END_REF].

Several points must be considered when choosing an animal model to study disease, preclinical testing or new therapeutic approaches for the disease: (1) the most important consideration is that the animal model must present the same genetic basis as the human disease; (2) the main hallmarks of the disease must be present in the model. In the case of DMD, hallmarks include muscle loss and weakness, respiratory insufficiency, cardiomyopathy and histological changes in skeletal muscle (e.g. centralized nuclei, fiber size variations) [START_REF] Torres | The mutant mdx: inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates[END_REF]; ( 3) the progression of the disease of this animal model must be well characterized; (4) the reproducibility of the phenotype and the facility to handle and maintain in standard laboratory housing conditions.

Murine models are among the most common in laboratories worldwide. These main models will be commented in the section below.

a. mdx mouse

The mdx mouse is naturally occurring from the C57BL/10-ScSn mouse line and presents a few genetic and biochemical similarities to the DMD genotypes/phenotypes [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF]. Mdx mice carry a point mutation in the exon 23, creating a premature stop codon that leads to the disruption of fulllength dystrophin. This model displays some of the main histological fibers hallmarks of DMD: size variation, necrosis, regeneration, central nuclei and revertant. However, this model present moderate muscle pathology and less compromised mechanical function, resulting in an almost normal lifespan. Studies shows that mdx mice present specific learning and memory deficits compared to C57BL/10 control mice [START_REF] Vaillend | Influence of dystrophin-gene mutation on mdx mouse behavior. I. Retention deficits at long delays in spontaneous alternation and bar-pressing tasks[END_REF]. Memory impairment was also reported in a fraction of DMD patients [START_REF] Billard | Cognitive functions in Duchenne muscular dystrophy: a reappraisal and comparison with spinal muscular atrophy[END_REF][START_REF] Bresolin | Cognitive impairment in Duchenne muscular dystrophy[END_REF].

Even though this model presents with mild phenotype, the nature of the mutation makes it a valuable model for testing potential treatments for DMD.

b. Double knockout dystrophin/utrophin (dKO or mdx/utrn -/-)

It is thought that mdx pathology is moderated by the partial functional replacement of dystrophin by an homologous protein called utrophin [START_REF] Rybakova | Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle[END_REF]. Utrophin presents similar size and functional domain organization to the dystrophin, with a shorter ROD domain (395kDa utrophin/427kDa dystrophin) and in physiological conditions is located at the neuromuscular junctions [START_REF] Nguyen | Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines[END_REF]. To overcome the weak points of the mdx model, a most severe model was generated by crossing C57BL/10ScSn-mdx mice to Utrntm1Ked mice [START_REF] Deconinck | Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy[END_REF]. Both mdx and the utrophin knockout mice show normal phenotype. Nevertheless, the double knockout dystrophin/utrophin mice present a much more severe phenotype with age, and die prematurely around 20 weeks of age. The absence of both dystrophin and utrophin have a higher impact in muscle integrity and leads to growth retardation, weight loss, spinal curvature and joint contractures, early diaphragmatic pathology and premature death [START_REF] Deconinck | Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy[END_REF][START_REF] Grady | Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy[END_REF].

c. mdx52

Because the mutation of the mdx mouse does not exactly correspond to DMD human mutations, Akari and colleagues generated the mdx52 mice. Bearing a mutation in exon 52, these mice present complete lack of dystrophin [START_REF] Araki | Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy[END_REF]. The disruption of exon 52 was induced to mimic the DMD phenotype, since the hot spot for human mutations is located around this exon. In addition to the lack of full-length dystrophin, the shorter isoforms Dp140 and Dp260 were also absent in this model. The skeletal muscle showed macroscopic hypertrophy andthe histology displayed variations in the fiber size, necrosis and regenerating process.

It is therefore considered to represent another good model mouse for DMD.

d. Dmd-mutated rats

Derived from the Wistar-Imamichi strain, the Dmd-mutated rats were created with the CRISPR Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats associated to Cas9) technology, introducing deletions in exons 3 and 16. Most of the DMD generated rats displayed absence or low levels of dystrophin. These animals also displayed degenerative lesions, infiltration of inflammatory cells, fibrosis in skeletal, diaphragm and cardiac muscles, centrally nucleated myofibers and difference in myofiber size. In general, the phenotype of the Dmd-mutated rats is more aggressive than mdx mice. This model presents a progressive cardiomyopathy and fibrosis, along with fat tissue infiltration. This represents an important model for testing new therapies for DMD, especially concerning the cardiac impact of the therapy [START_REF] Larcher | Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy[END_REF][START_REF] Nakamura | Generation of muscular dystrophy model rats with a CRISPR/Cas system[END_REF].

e. Canine model (GRMD)

The model that better mimics the human pathology is the Golden Retriever Muscular Dystrophy (GRMD) dog. The GRMD dog is a natural occurring model that bears a point mutation in the splice acceptor site of intron 6, causing a shift in the reading frame due to complete deletion of exon 7 from the mature mRNA.

Unlike the dystrophin-deficient mdx mouse, which presents a mild phenotype, the GRMD dogs develop a progressive and fatal disease similar to the human condition with growth retardation and progressive muscle atrophy. In addition, the absence of dystrophin in these dogs have a particular impact in the pharyngeal muscles, which leads to excessive drooling [START_REF] Ambrósio | Identification of three distinguishable phenotypes in golden retriever muscular dystrophy[END_REF][START_REF] Shimatsu | Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ[END_REF][START_REF] Valentine | Canine Xlinked muscular dystrophy. An animal model of Duchenne muscular dystrophy: clinical studies[END_REF]. . Cardiomyopathy is common and usually leads to cardiac failure [START_REF] Kornegay | Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies[END_REF]. Even though this model is the best suited for preclinical testing, the main disadvantage of this model is the high costs of maintaining lab facilities able to house these large animals.

f. Pig model

A new DMD model was created in 2016 with the CRISPR-Cas9 technology. The porcine model was created by injecting Chinese Diannan miniature pig's zygotes with CRISPR tools targeting the dystrophin exon 27. The targeting efficacy of this method was 50%, with considerable dystrophin decrease in several tissues [START_REF] Yu | Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy[END_REF]. Further histological, behavioral and pathophysiological analyses must be performed to confirm if this model is capable of mimicking the DMD phenotype in humans. One of the main interest of a porcine model when researching new therapeutic approaches is the similarity between the pig and the human physiology.

g. Other models

The hypertrophic feline muscular dystrophy (HFMD) displays a mild pathology, similar to mdx mouse. The HFMD mutation is deletion of exon 1, leading to the absence of all full-length dystrophin isoforms. The skeletal muscle of the HFMD cat undergoes repeated cycles of degeneration and regeneration but does not develop the debilitating fibrosis that is characteristic of DMD phenotype. The most obvious features of HFMD muscle is cardiac enlargement and marked hypertrophy starting at 9 months of age, which can ultimately lead to lethal oesophageal obstruction and kidney failure [START_REF] Gaschen | Changes of skeletal muscle in young dystrophindeficient cats: a morphological and morphometric study[END_REF][START_REF] Gaschen | Cardiomyopathy in dystrophin-deficient hypertrophic feline muscular dystrophy[END_REF][START_REF] Kohn | Muscular dystrophy in a cat][END_REF]. The HFMD cat is not widely used as a model because it combines the practical disadvantages of being a large and emotive animal, with the scientific objection of having limited pathological similarity to DMD.

III.2 Therapeutic approaches for Duchenne muscular dystrophy using recombinant Adeno-associated virus

Effective gene-transfer strategies must ensure the transfer of the gene to a cell population, where it should function to correct the abnormal phenotype. Gene transfer for a hereditary disorder can be achieved either by ex vivo or in vivo gene transfer.

III.2.1 Mini-& micro-dystrophin strategies

The in vivo allele complementation is the most direct strategy for gene transfer.

Human in vivo studies for monogenic disorders have administered a vector containing the therapeutic DNA either directly to the organ of interest or into the blood stream.

For DMD, observations of the milder BMD phenotype lead scientists to attempt to restore a truncated form of dystrophin. Direct transfer of full-length or truncated forms of dystrophin (mini-dystrophin, a 6.3kbp cDNA and micro-dystrophin genes of 3.8kbp coding for N-and C-terminal ends of dystrophin) were performed in several animal models of DMD (figure 16).

First attempts to deliver the dystrophin gene by using non-viral vectors were not satisfactory [START_REF] Acsadi | Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs[END_REF]. Even though this method may reach a high number of newly expressed dystrophin, the distribution is not equal resulting in a mosaic pattern not able to protect the fibers that will eventually break and undergo degeneration-regeneration cycles [START_REF] Vilquin | Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies[END_REF].

Because of its small packaging capacity, AAV is only able to deliver the short dystrophin isoforms. Several studies used this viral vector to successfully deliver microdystrophin [START_REF] Harper | Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy[END_REF] and mini-dystrophin [START_REF] Wang | Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model[END_REF][START_REF] Watchko | Adeno-Associated Virus Vector-Mediated Minidystrophin Gene Therapy Improves Dystrophic Muscle Contractile Function in mdx Mice[END_REF]. They achieved similar results rescuing the normal muscle fiber morphology and membrane integrity, reversing the mdx phenotype and improving contractile functions. For example, one study shows that micro-dystrophin cDNA gene construct of approximately 3.8 kb was introduced into the skeletal muscle of 12-day-old nude/mdx mice using AAV vectors, resulted in specific sarcolemmal expression of micro-dystrophin in more than 50% of myofibers , and effective restoration of the DAPC components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology [START_REF] Dickson | Recombinant micro-genes and dystrophin viral vectors[END_REF][START_REF] Fabb | Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice[END_REF]. the presence of a complementary sequence to the histone downstream element (HDE). The presence of a hairpin structure at the 3' end of the U7snRNA offers stabilization and the exposed 5'end of the U7snRNA (Figure 17) [START_REF] Imbert | Viral Vector-Mediated Antisense Therapy for Genetic Diseases[END_REF][START_REF] Schümperli | The special Sm core structure of the U7 snRNP: farreaching significance of a small nuclear ribonucleoprotein[END_REF].

The Sm core of the U7snRNP consists of seven proteins: Lsm10, Lsm11, B/B' (alternative splicing products), D3, E, F and G disposed in an organized structure.

Modifications in the U7 Sm binding site by the replacement of a consensus sequence derived from the spliceosomal snRNPs (U7SmOPT), implicates the accumulation of this complex in the nucleus instead of the cytoplasm. Moreover, this modification leads to the inability to cleave the histone pre-mRNA target due to the replacement of Lsm 10 and 11 by D1 and D2 Sm proteins [START_REF] Grimm | The low abundance of U7 snRNA is partly determined by its Sm binding site[END_REF][START_REF] Stefanovic | Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes[END_REF]. In addition, the modification of the HDE sequence by an antisense sequence can be used to induce the retargeting of the splicing modulation [START_REF] Schümperli | The special Sm core structure of the U7 snRNP: farreaching significance of a small nuclear ribonucleoprotein[END_REF].

The modified U7 snRNA can also carry a second antisense sequence to target exonic splicing enhancer/silencer (ESE/ESS) factors, which can optimize the effect of exon skipping or exon re-inclusion. Altogether, these findings suggest the applicability of U7snRNAs as a tool for pre-mRNA splicing modulation for (1) ability to accumulate in the nucleus where the spliceosome machinery takes place;

(2) capacity to carry antisense sequences to specific target sites.

Another great advantage of the U7snRNA system is that its small size offers the possibility of vectorization by several types of viral vectors, in which several cassettes can be included in the same viral genome.

The U7snRNA technology was first tested in an in vitro model of β-thalassemia and results showed that the use of a U7snRNA carrying an antisense sequence targeting this mutation corrected more than 50% of aberrant splicing and subsequent re-expression of full-length β-globin protein [START_REF] Gorman | Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs[END_REF]. Since this development, U7snRNA have been successfully exploited to correct mutations in a number of inherited diseases, including DMD.

2008). Restoration of dystrophin expression in cardiac muscle has also been

demonstrated in a large animal model, the golden retriever muscular dystrophy dog (GRMD), using percutaneous transendocardial delivery of AAV-U7 snRNA [START_REF] Bish | Long-term Restoration of Cardiac Dystrophin Expression in Golden Retriever Muscular Dystrophy Following rAAV6-mediated Exon Skipping[END_REF]. Additional studies from Garcia and Moullier have shown correction of the dystrophic phenotype and partial recovery of muscle strength after intramuscular (IM) and IV injections of AAV-U7 snRNA in GRMD dogs (Le [START_REF] Guiner | Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients[END_REF][START_REF] Vulin | Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping[END_REF]. Altogether these results have advanced the feasibility of exon-skipping in different animal models, suggesting promising therapeutic outcomes based on AAV-snRNA. Consequently, different groups have tried to identify the most appropriate antisense sequences targeting the human dystrophin pre-mRNA that are able to induce the highest level of skip. Several groups have achieved skipping of exon 51 in human cells carrying deletions of exons 48-50 or 45-50 using U1 snRNA [START_REF] Benchaouir | Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice[END_REF][START_REF] Incitti | Exon Skipping and Duchenne Muscular Dystrophy Therapy: Selection of the Most Active U1 snRNA Antisense Able to Induce Dystrophin Exon 51 Skipping[END_REF].

Appreciating that DMD is caused by mutations at different gene loci and that 70% are located between exons 45 and 55, Goyenvalle et al. have designed eleven U7 snRNAs targeting these different human dystrophin exons. They have provided very encouraging results in DMD-patient myoblasts and demonstrated a multiple skipping of at least 3 exons with a single vector in a human DMD transgenic mice (hDMD) [START_REF] Goyenvalle | Engineering Multiple U7snRNA Constructs to Induce Single and Multiexon-skipping for Duchenne Muscular Dystrophy[END_REF]. Our group has also investigated the use of a 'bifunctional U7snRNA' carrying a tail harbouring binding sites for a splicing silencer (hnRNPA1) in addition to the antisense sequence [START_REF] Goyenvalle | Enhanced Exon-skipping Induced by U7 snRNA Carrying a Splicing Silencer Sequence: Promising Tool for DMD Therapy[END_REF].

Percutaneous transendocardial delivery of recombinant AAV6 [START_REF] Bish | Long-term Restoration of Cardiac Dystrophin Expression in Golden Retriever Muscular Dystrophy Following rAAV6-mediated Exon Skipping[END_REF], and AAV8 injected by locoregional transvenous perfusion of the forelimb (Le [START_REF] Guiner | Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients[END_REF] were performed. A 5-year follow-up ruled out immune rejection but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes, suggesting that repeated administration may still be required [START_REF] Vulin | Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping[END_REF]. The production of neutralizing antibodies following the first injection of AAV prohibit repeated injections of this type of vector as discussed later. To circumvent this issue and improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months [START_REF] Peccate | Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles[END_REF].

b. Meganucleases

Meganucleases (MN) were the first genome editing tool used to correct mammalian cells [START_REF] Choulika | Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae[END_REF][START_REF] Cohen-Tannoudji | I-SceI-induced gene replacement at a natural locus in embryonic stem cells[END_REF]. This tool has been used twice in the correction of out-of-frame dystrophin. The first study cotransfected 293FT cells, a fast growing human embryonic kidney cell line that stably express the SV40 antigen, with a plasmid coding for the dog mini-dystrophin and a plasmid coding for an engineered MN to target this specific mutation. Results

showed that MN introduced internal deletions (indels), restoring the reading-frame with high efficiency [START_REF] Chapdelaine | Meganucleases can restore the reading frame of a mutated dystrophin[END_REF]. The second study used this tool to correct DMD patient's myoblasts carrying exons 45-52 deletions. In this work, the co-transduction of a MN targeting exon 44 and a homologous recombination repair matrix, both vectorized by integration-competent lentiviral vectors, was able to generate a corrected full-length dystrophin [START_REF] Popplewell | Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in[END_REF]. This strategy is not widely used today specially for the difficulty in optimizations that allows specific targeting, therefore low specificity that leads to toxicity due to off-site cleavages [START_REF] Bire | Combining long-term expression and secureintegration by specific locus targeting: Old solutions and new perspectives[END_REF]. In addition, one study in particular show that high doses lead to lethality in mice [START_REF] Gouble | Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break[END_REF].

c. Zinc-Finger Nucleases

ZFN are an important gene editing tool for its versatility and capacity to recognize long target sequences, which can be useful when targeting eukaryotic cells. In addition, the possibility to engineer new sequences confers versatility to this tool [START_REF] Urnov | Genome editing with engineered zinc finger nucleases[END_REF]. ZFN are composed by Zinc-finger proteins (ZFP) able to bind DNA combined to a FokI restriction enzyme, capable of inducing a DSB. This tool can be used in order to induce gene disruption, gene addition or homology-directed recombination. For DMD, two ZFN have been used to flank exon 51 and restore the reading frame of the dystrophin mRNA. These tools were electroporated in vitro into human DMD myoblasts generating a population producing a truncated functional dystrophin. These cells were then transplanted in vivo into immunodeficient mice and resulted in new dystrophin expression. ZFN presented some cytotoxicity in human cells that was previously described and offtarget activity was 25% higher than on-target activity (Ousterout et al., 2015a).

Gene editing mediated by ZFN became an important tool in the generation of transgenic animal models. However, the ZFN methodology presents high costs and may not be easily implanted in standard molecular biology laboratories [START_REF] Fernandez Alanis | An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects[END_REF].

d. Transcription activator-like effector nucleases (TALENS)

The TALEN technology was developed in the last decade and is composed by non-specific DNA-cleaving nuclease combined to a DNA-binding domain.

TALENs, much as ZFN, are fusion proteins carrying a catalytic domain formed by the presence of the endonuclease FokI. In addition, the presence of a designed TALE DNA-binding domain can be easily engineered to carry sequences specific to the target gene [START_REF] Ousterout | Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients[END_REF]. Compared to ZFN, this strategy presents less off-target effects, higher safety, higher efficacy easier to engineer [START_REF] Joung | TALENs: a widely applicable technology for targeted genome editing[END_REF]. This technology was already applied to a number of animal models such as zebrafish [START_REF] Bedell | In vivo genome editing using a highefficiency TALEN system[END_REF], rats [START_REF] Tesson | Knockout rats generated by embryo microinjection of TALENs[END_REF], pigs [START_REF] Carlson | Efficient TALEN-mediated gene knockout in livestock[END_REF], cows [START_REF] Carlson | Efficient TALEN-mediated gene knockout in livestock[END_REF] and in the editing of human somatic [START_REF] Cermak | Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[END_REF][START_REF] Miller | A TALE nuclease architecture for efficient genome editing[END_REF][START_REF] Reyon | FLASH assembly of TALENs for high-throughput genome editing[END_REF] and pluripotent stem cells [START_REF] Hockemeyer | Genetic engineering of human pluripotent cells using TALE nucleases[END_REF].

TALENs have been engineered to target the exon 51 of the dystrophin gene and promote the restoration of the reading frame following excision of this exon. The transfection of the tools was accessed by electroporation to increase the efficacy rate. This promoted the restoration of dystrophin expression in vitro in DMD skeletal myoblasts and dermal fibroblasts by NHEJ. The efficacy of dystrophin correction however was very low, with only 5-7% alleles presenting the internally truncated new dystrophin [START_REF] Ousterout | Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients[END_REF].

e. CRISPR-Cas9

The recent advent of the CRISPR-Cas9 technology, powerful and revolutionary genomic editing tool, marks a milestone in the context of therapeutic approaches to genetic diseases. This system is a RNA-guided, nuclease-mediated genome editing tool derived from bacterial immune system. The nuclease Cas9 binds to the target DNA next to the protospacer adjacent motif (PAM) guided by the single-guide RNA (sgRNA), and generates DSB. The DSB can be repaired either by nonhomologous end-joining (NHEJ), which leads to insertion/deletion (indel) mutations, or by homology-directed repair (HDR), which requires an exogenous template and can generate a precise modification at a target locus (Figure 19). By judicious selection of a guide RNA sequence, the enzyme Cas9 can indeed bind the target DNA sequence (link guide RNA to target DNA) and induces a double-strand break at a specific site. Previously, the Cas9 was extracted from Streptococcus pyogenes (SpCas9) and presented a large size not being able to be packaged by some viral vectors as AAV. Recently, one group described six smaller bacterial Cas9 skeletal muscle cells and successfully detected the expression of full-length dystrophin protein, normalized calcium spark activity and the restoration of the DAPC [START_REF] Li | Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9[END_REF]. Ousterout et al and Iyombe-Engembe et al targeted the hotspot exons in DMD patient myoblasts. The former study designed several sgRNAs to target exons 45-55. Following in vitro restoration of the dystrophin, these myoblasts were transplanted into immunodeficient mice. This strategy could target up to 62% of all DMD mutations (Ousterout et al., 2015b); the latter generated several sgRNAs to target exons 50-54 in DMD patient myoblasts in vitro and in the mdx mutation in vivo. They were able to restore the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdxmice [START_REF] Iyombe-Engembe | Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method[END_REF].

Following studies applied the technology to correct DMD/mdx mutations by NHEJ and HDR. One group published two works, one in which they attempt to correct the mutation in the germline of mdx mouse [START_REF] Long | Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA[END_REF] and the other in which they injected CRISPR-Cas9 tools, also in mdx mice, vectorized by AAV9 at P1 (postnatal day 1) (intraperitoneally), P12 (postnatal day 12) (intramuscularly) and at P18 (postnatal day 18) (by retro-orbital injection) [START_REF] Long | Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy[END_REF]. In the first study the Cas9 enzyme, the sgRNA, and the HDR template were injected into mouse zygotes to correct the disease-causing gene mutation in the germ line.

Results showed that in seven mice the HDR occurs, however in four of them the correction was made by NHEJ. In addition, muscles displayed a mosaic pattern of dystrophin restoration and efficacy varied between 2 and 100% of dystrophin positive fibers. Because HDR does not occur with high efficacy in post-mitotic cells, they attempt to induce correction by NHEJ in neonatal mice with injection of a sgRNA and Cas9 enzyme. This resulted in approximately 80% of exon 23 exclusion in mdx mice. In accordance with the first study, no off-target effects took place [START_REF] Tabebordbar | In vivo gene editing in dystrophic mouse muscle and muscle stem cells[END_REF]. Tabebordbar et al. also used AAV9 to vectorize CRISPR-Cas9 and the sgRNA guides to mdx mice [START_REF] Tabebordbar | In vivo gene editing in dystrophic mouse muscle and muscle stem cells[END_REF]. Nelson et al. used AAV8 to deliver CRISPR-Cas9 system by intramuscular and systemic injections in neonatal mdx mice [START_REF] Nelson | In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy[END_REF]. In conclusion, these three groups were able to restore dystrophin by NHEJ and reduce pathology in this animal model of DMD.

Bengsston et al used a single (Cas9/sgRNA) or dual (Cas9/sgRNA with dystrophin homology sequence) AAV-mediated CRISPR muscle specific-Cas9 systemic delivery into the mdx 4cv , an immunodeficient dystrophic mouse model of DMD. They were able to target both skeletal and cardiac muscle with a widespread expression of dystrophin that reached up to 70% positive fibers in myogenic area and have increased the muscle force [START_REF] Bengtsson | Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy[END_REF]. In addition, using a new class of CRISPR-Cas9, named Cpf1 (CRISPR from Prevotella and Francisella 1), one group was the first to apply this technology forDMD . They were able to correct the DMD mutation in iPSCs cells and mdx mice by both NHEJ and specially by inducing HDR. Not only the full-length dystrophin was reexpressed in these animals, but the pathophysiological hallmarks were rescued [START_REF] Zhang | CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice[END_REF].

Altogether these results show a clear evolution in the treatment of DMD by the CRISPR-Cas9 system. The increasing importance of viral vectors in the practical application of gene transfer demands the improvement of production processes, especially with a view to enhance the yields and purity of the final product.

OBJECTIVES & METHODS

OBJECTIVES

The first specific objective is to improve the AAV production procedure in order to have optimal titers of recombinant AAV vectors for in vivo treatments. Our group showed that the quantity of viral vectors injected per body mass is a determinant factor to achieve long-term therapeutic efficacy (Le [START_REF] Hir | AAV Genome Loss From Dystrophic Mouse Muscles During AAV-U7 snRNA-mediated Exon-skipping Therapy[END_REF].

With this aim we developed new techniques for AAV purification for both research and pilot scale that allows the increase of viral vector concentration and an optimization of production time and costs.

Small-scale productions are usually performed through the classical tritransfection method in adherent human embryonic kidney 293 (HEK293) cell line.

Optimization of downstream technologies in this last decade led to better yields and higher levels of vector purity. Generally, vectors are extracted from the cell pellet following a 72h culture in a cell culture plate [START_REF] Grieger | Production and characterization of adenoassociated viral vectors[END_REF] or from the supernatant [START_REF] Doria | AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently[END_REF][START_REF] Lock | Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale[END_REF][START_REF] Vandenberghe | Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing[END_REF]. For large-scale rAAV productions, technological improvements in methods using baculovirus/insect cell system confers the possibility of scaling up the levels of production much more than can be provided by processes using tri-transfection in HEK293T adherent cells. This is a particularly advantageous characteristic for laboratories involved in pre-clinical studies in large animals and clinical trials.

During my thesis, we were interested in developing a new simple procedure capable of improving the purification methods for rAAV vectors. In addition, improvement in production yields and reduction in time spent for purification were important parameters evaluated in this new method.

II. Gene vector application: gene editing for DMD

Recent developments of the CRISPR-Cas9 bacterial system to perform gene editing in eukaryotic cells has led us to evaluate this approach in the murine DMD model, the mdx mouse. Using this system, several groups achieved to generate the re-expression of a truncated, but functional, dystrophin isoform by NHEJ in patient myoblasts and in the mdx mice [START_REF] Bengtsson | Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy[END_REF][START_REF] Iyombe-Engembe | Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method[END_REF][START_REF] Long | Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA[END_REF][START_REF] Long | Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy[END_REF][START_REF] Nelson | In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy[END_REF]Ousterout et al., 2015b;Tabebordbar et al., 2016;[START_REF] Xu | CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice[END_REF]. As mentioned before, the truncated dystrophin is less stable than the full-length isoform and still characterizes a dystrophic phenotype.

It is in this context that our goal is to correct the mdx mutation by HDR induced repair mediated by the CRISPR-Cas9 system. To this end we designed two plasmids: one carrying the Cas9 enzyme and a sgRNA able to recognize the PAM sequence adjacent to the mdx mutation; the second plasmid carries a template with the corrected sequence to correct the mutation. These plasmids were used in in vitro experiments on primary mdx myoblasts and vectorized by AAV9 for in vivo experiments in the mdx mouse model. Here will be presented preliminary results of this application.

MATERIAL AND METHODS

Below I described a simplified methodology for this work. For a more detailed methodology consult the pages of the associated articles.

I. Methodology

Upstream process

The upstream process was carried at the same way for all three cultures.

HEK293 were seeded 3 days previous the transfection in a ten-layer cell culture plate (HyperflaskÒ). Tri-transfection was performed with polyethylenimine (PEI) (Sigma-Aldrich, France) mixed with pAdDeltaF6 helper plasmid, pAAV2/9 packaging plasmid, and the pAAV-mSEAP plasmid. Transfected cells are incubated for 72 hours at 37 °C under 5% CO2.

Downstream process

The cell pellet (CP) and supernatant (SN) extraction method starts by mechanical shaking of the HyperflaskÒ until cells detach. The whole culture medium is centrifuge to separate the cell pellet from the supernatant and subsequent steps take different paths: CP is treated with lysis buffer and subsequent heat shock lysis. The SN is clarified by centrifugation, nuclease treatment and filtration.

The new protocol (TC for total culture) key step combine the treatment of the cell pellet and supernatant. The whole culture is treated with Triton X-100 non-ionic detergent and RNase A for 1h at 37°C under shaking. The clarification process is done by centrifugation and filtration through a 0.45 µm filter. Tangential flow filtration is performed to concentrate both SN and TC production from the initial volume of 1L to approximately 35mL. The following steps remain the same for all three purification methods. Following these steps, all three methods are purified using a isopycnic iodixanol gradient combined with ultracentrifugation, and diafiltration.

In vitro experiments: HEK 293 T cells were transduced with vectors derived from the three methods at multiplicity of infection (MOI) of 10 5 . Two days' posttransduction, the culture medium is recovered and cells are fixed. mSEAP expression can be measured both in the supernatant from the cell culture and following fixation of cells with commercial kits detailed in the protocols (appendix).

Upstream process

Two BIIC systems carrying reporter genes were generated. The first coding for the green fluorescent protein (GFP) and a second one coding for the mSEAP.

Non-adherent Sf9 insect cells were cultivated in serum-free medium following two conditions: research scale (2L) in erlenmeyer vessels and pilot scale (5L) in the "wave-type" bioreactors both at 26°C under shaking. Sf9 cells were infected with BIIC at a ratio of 1 to 10 4 (v/v BIIC vs Sf9) and culture was ended 72h post-infection.

Downstream process

The downstream process begins with the clarification of the suspensions with Triton X-100 and nuclease treatment, followed by filtration through a 0,45 µm dead end filter for both research and pilot scale cultures. The clarified lysate is concentrated by tangential flow filtration to reach about 20× concentration factor.

The key step to increase the purification efficiency is the isopycnic iodixanol gradient ultracentrifugation. Previously, with the four layer iodixanol gradient, only 16mL of lysate could be purified at a time. With an adaptation of this gradient, now we have three phases that allows the purification of almost double (30mL) of vector suspension, increasing the final yields. In addition, the recovery of 4,5mL instead of 6mL decreased the time of diafiltration.

In vitro experiments HEK 293 T cells were transduced at a logarithmic MOI ranging from 10 to 10 4 by rAAV9-GFP. 48h later, GFP expression was analyzed qualitatively in some wells by an inverted fluorescent Leica microscope, and quantitatively by flow cytometry.

In vivo experiments

Two injection protocols were designed: an intramuscular protocol in which adult C57BL/10 mice TAs were injected with a total of 2.0 × 10 12 AAV2/9-mSEAP vectors from the new purification method, and an intravenous protocol through retro-orbital delivery of 1.0 × 10 13 vg per gram of body mass of the same vector.

Controls were injected with the same volume of PBS (100µL for IV and 50µL for IM). Blood was drawn weekly for four weeks to measure mSEAP activity in the serum (protocol in appendix). After the sacrifice, TAs were harvested from the IM group and several organs (heart, diaphragm, liver, kidneys, brains, and skeletal muscles -TA, gastrocnemius, quadriceps, triceps) were harvested from the IV group. mSEAP staining was performed following preliminary fixation of tissues (protocol in appendix).

III. Gene vector application

In vitro experiments

In vitro experiments were performed used primary mdx myoblasts and C2C12 cells. Around 24h post-seeding, when cells reached 60-70% confluency, myoblasts were transfected with both plasmids at different ratios 1:1 1:2 and 1:4 (crmdx : crmdxD760). 72h post-transfection cells were harvested and genomic DNA extracted.

In vivo experiments

In vivo experiments were performed in adult male C57BL/10 mice. 72h and 96h

following Cardiotoxin injections, mice were injected with rAAV9 at a ratio of 1:4

(crmdx : crmdxD760). Mice were sacrified 10 days post-injection and muscles were harvested.

Analysis

Genomic DNA was extracted using Macherey NagelÒ genomic DNA extraction kit. PCR amplification was performed to observe the wt/mdx band using primers Fs + Rwt-mdx, and the HDR band with primers Fs + Rcr (See Figure 37 for primers sequence). Bands were separated in an agarose gel and sequencing was performed by GATC Biotech.

I. ARTICLE 1: Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adenoassociated virus vectors

Authors: Dias Florencio G, Precigout G, Beley C, Buclez PO, Garcia L, Benchaouir R

Introduction

Recombinant adeno-associated viruses (rAAV) are used as tool for gene transfer applications. Clinical trials, using rAAV as gene therapy vectors, have yielded promising results [START_REF] Kotterman | Engineering adeno-associated viruses for clinical gene therapy[END_REF] and first marketing approval, using a rAAV1 vector, was delivered by the European Union in October 2012 for the treatment of familial lipoprotein lipase deficiency [START_REF] Gaudet | Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial[END_REF][START_REF] Salmon | Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera ® )[END_REF]. These vectors belong to the Parvoviridae family, nonenveloped viruses embedding a single-stranded DNA genome packaged into a 25 to 30 nanometer icosahedral shaped capsid. These last two decades offered large panel of upstream and downstream methods for rAAV, aimed at constantly improving the yields, the purity, but also the scale and the time of the processes [START_REF] Ayuso | Production, Purification and Characterization of Adeno-Associated Vectors[END_REF]. Among them, tri-transfection remains the most popular method of rAAV production for a large number of research laboratories [START_REF] Grimm | Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors[END_REF][START_REF] Salvetti | Factors Influencing Recombinant Adeno-Associated Virus Production[END_REF][START_REF] Xiao | Production of high-titer recombinant adenoassociated virus vectors in the absence of helper adenovirus[END_REF]. This transient approach uses transfection of the human embryonic kidney 293 (HEK293) cell line with three different plasmids. The first harbors the transgene of interest flanked by the viral inverted terminal repeat (ITR) sequences (essential elements for genome replication and packaging), the rep and cap genes coding plasmid (for the expression of viral enzymes and structure proteins respectively) and the adenoviral helper coding plasmid (expressing the genome replication helping functions). The adherent cells can grow in many precoated tissue culture plates commercially available, offering multitude of surface areas, and giving the possibility to scale up the levels of production [START_REF] Liu | Optimized production of high-titer recombinant adeno-associated virus in roller bottles[END_REF]. Tri-transfected HEK293T cells rapidly initiate rAAV production, generally ending culture within 72 hours posttransfection, and vectors are usually recovered from cell lysate after elimination of medium [START_REF] Grieger | Production and characterization of adenoassociated viral vectors[END_REF]. Chemical and/or physical treatments are required to release the vectors from the cells, followed by microfiltrations through specific membranes. After these clarifications steps, downstream processes can be performed by different methodologies. Among them, density gradient ultracentrifugation remains the most used method for research grade rAAV purification. Iodixanol-based isopycnic ultracentrifugation is generally preferred to the classical CsCl2 approach due to its higher particles recovery yields [START_REF] Hermens | Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System[END_REF][START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. Chromatographic methods, using either ion-exchange [START_REF] Okada | Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes[END_REF][START_REF] Qu | Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography[END_REF][START_REF] Urabe | Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression[END_REF] or affinity-based approaches are also used [START_REF] Anderson | A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction[END_REF][START_REF] Auricchio | Isolation of Highly Infectious and Pure Adeno-Associated Virus Type 2 Vectors with a Single-Step Gravity-Flow Column[END_REF][START_REF] Koerber | Engineering Adeno-Associated Virus for One-Step Purification via Immobilized Metal Affinity Chromatography[END_REF]. However, unlike ultracentrifugation, chromatographic techniques are more serotype specific and need more technical adjustments to separate the full particles from empty particles.

It is surprising to note that, up to recently, only few studies cared to verify the amount of rAAV into the supernatant. Recent developments described the possibility to recover the viral vectors from the culture medium, avoiding the more complex and time-consuming process issue from the treatment of the cellular fraction [START_REF] Doria | AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently[END_REF][START_REF] Lock | Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale[END_REF][START_REF] Vandenberghe | Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing[END_REF]. Moreover, the rAAV collected from the conditioned medium is still functional after in vivo delivery, despite the probable presence of proteases liberated by dead cells during production time lapse.

Nevertheless, in both cases (cells versus supernatant processing), a significant amount of rAAV is inevitably lost, reducing the absolute number of vector particles to be recovered. One obvious method could be to treat both cell pellet and supernatant in parallel and pool batches after purification. However, this method will considerably increase the time of processing and eliminate the benefice brought by the simplest and faster supernatant purification method.

Here we show that the whole culture, including cells and supernatant, can be entirely processed using a very simple detergent treatment during the initial clarification step. This new method leads to higher rAAV recovery without diminishing the simplicity and fastness of the purification process. A first concentration step is performed by using a simple tangential flow filtration method, and is followed by an isopycnic density gradient. The entire process leads to high vector recovery and purity, and allows notable reduction of process duration.

Moreover, the rAAV conserve efficient in vitro and in vivo functionality that surprisingly appears better than vectors purified by classical methods. This new technological approach could be easily implemented for higher production scales performed in large cell factory systems, and advantageously bridges research and pilot development activities on rAAV gene therapy vectors.

Results

Nonionic detergent action highly simplifies rAAV purification process

Original rAAV purification process from tri-transfected HEK293T cells has recently evolved by using supernatant rather than cell pellet to recover viral particles [START_REF] Doria | AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently[END_REF]. The authors concluded that purification from the medium drastically diminishes the process duration compared to classical AAV harvesting from the cell pellet. Nevertheless, the sidelining of vectors encased into the cells seemed to us disadvantageous in term of yields and particle recoveries. We thus proposed an efficient alternative process based on vector recovery from the whole culture, summing particles from both cells and supernatant. As described in Figure 20a, we compared three rAAV downstream processes issued from tri-transfection of HEK293T cells cultivated in Hyperflask vessels. In each independent experiment (n = 4), two Hyperflasks were transfected from same transfection mixture to avoid any experimental bias due to plasmid quality or quantity. 560 ml of medium was used to completely fill one Hyperflask. The first vessel was used to recover rAAV from cell pellet (CP) after mechanical cell detachment (manual tapping and shaking) followed by a low-speed centrifugation (500 × g) to avoid physical cell lysis. The resulting supernatant (SN) was treated independently, while the second vessel was used to recover vectors from total cell culture (TC) after simple addition of Triton X-100 at 0.5% final concentration. Detergent and endonuclease (RNAse) treatments were performed at 37 °C for 1 hour under vigorous automatic Hyperflask shaking. One hour in presence of detergent was enough to completely lyse cells as determined by cell viability counting (not shown). In both cases, the original Hyperflask was washed once with 440 ml of phosphate buffered saline (PBS), meaning that 1 l of conditioned medium (SN or TC) was collected from this initial process. The clarification step showed clear difference in process duration with 5.5, 2, and 1.5 hours for CP, SN, and TC methods, respectively. Beyond this crucial parameter, it is important to note that the TC approach proposes a more ergonomic activity since it uses fewer stages before the 0.45 µm filtration process that ends all the clarification steps. In particular, the time consuming and complex heat-shock lysis method (3 hours), which comprises four sequential freeze-thaw cycles (-80 °C/37 °C), was suppressed from the SN and TC methods. Despite the concentration step performed for the SN and TC methods (Figure 20a), total process duration is significantly decreased compared to the CP method since only 6 hours are needed with TC approach to obtain highly purified vectors from one Hyperflask against 6.5 and 8.5 hours for SN and CP conditions respectively. This means that 1 day of downstream process is enough to recover highly purified AAV product. Beyond the aim for process simplification, we also pay special attention to the cost generated by our new experimental procedure. Indeed, we decided to omit the addition of DNase during the TC and SN clarification stages especially due to its prohibitive price. Surprisingly, this omission does not affect the purification process since we observed that the viscosity generated during concentration steps (tangential flow filtration (TFF)) was drastically reduced by simply adding RNAse (supplementary non-published material: Figures 25 and26).

Moreover, the use of an isopycnic density gradient at the end of the process, acting as a barrier for DNA sedimentation, allows removing of most of the soluble DNA in upper part of the gradient which becomes well separated from the bottomed collected fractions [START_REF] Dorin | Fast separations of plasmid DNA using discontinuous gradients in the preparative ultracentrifuge[END_REF].

TC method significantly increases the yields without altering final purity of rAAV preparations

To evaluate the effect of detergent treatment on AAV particles, we first performed a production of rAAV2/9-mSEAP vectors by using the tri-transfection protocol as described above and also in material and methods (Figure 21a). From four independent productions, we used quantitative polymerase chain reaction (Q-PCR) to titer viral genomes (vg) from CP, SN, and TC methods. We show that number of vg was significantly higher in TC-derived vectors than from CP or SN processes (Figure 21b and Supplementary Table 2). Quality of the final AAV product was also evaluated by silver staining of 10 10 vg issued from the three distinct processes which were deposited and migrated in parallel through sodium dodecyl sulfate 4/12% polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Figure 21c, no alteration of AAV capsids (VP1, VP2, and VP3) was observed in TC-derived vectors compared to the other methods (CP and SN). Moreover, capsids ratio was respected (about 1/1/10 for VP1/VP2/VP3 respectively). We conclude that the Triton X-100 treatment allows better rAAV recovery without altering the physical quality of viral vector particles. Quantification revealed that higher rAAV recovery is achieved for TC-derived vectors, with a twofold difference between TC and CP processes (P = 0.134) and fourfold significant difference (*) between TC and SN process (P = 0.037). Refer to Materials and Methods for quantification protocol and methods of statistical analyses. Refer also to Supplementary Table 2 for titration details. (c) AAV vector quality determined by silver staining of SDS-PAGE. 10 10 vg of rAAV from CP, SN, and TC processes are denatured, reduced, and migrated in 4%/12% SDS-PAGE. Silver staining of the gel reveals the three AAV capsid components materialized by 82 kD, 67 kD, and 60 kD bands, corresponding to VP1, VP2, and VP3 proteins respectively. Note that no other contaminating band can be distinguished on either side of the AAV capsid proteins. SM, size marker for molecular weight determination.

TC method does not alter the in vitro transduction capacity of rAAV vectors

To better evaluate a prospective effect of detergent treatment in AAV functionality, we performed an in vitro assay by transducing permissive HEK293T cells with an equal quantity of rAAV2/9-mSEAP vectors from CP, SN, or TC processes.

Because of the known low in vitro transduction efficiency of rAAV, we used a multiplicity of infection equal to 10 5 . Fourty-eight hours post-transduction, supernatants were collected and adherent cells were fixed for mSEAP activity revelation. Phase microscopy pictures reveal no general differences in mSEAP activity between CP-, SN-, or TC-derived vectors (Figure 22a). However, at lower magnification, we observed more product deposits in TC cell culture when compared to SN and at less extent when compared to CP (Supplementary Table 2). To verify these findings, we quantified the mSEAP activity in supernatants derived from these previously transduced cell cultures (Figure 22b). In accordance, we observed higher mSEAP activity in cultures transduced from TC derived rAAV with a highly significant difference (P < 0.01) between transduction from SN and TC vectors (66.88 ng/ml ± 0.85 and 162.03 ng/ml ± 1.84 respectively). These results suggest that TC method may better conserve high rAAV quality, increasing the amount of functional vectors. 3 for more details.

rAAV vectors issued from TC method perform a better in vivo transduction

Beyond the in vitro proof of concept, we decided to evaluate the in vivo functionality of our rAAV vectors. Intramuscular delivery of CP-, SN-, or TCderived vectors was performed in C57BL/10 adult mice. Two different doses were tested in order to select non saturating mSEAP concentrations and to allow better comparison of transduction efficiency (Figure 23b and Supplementary Figure 24b). Suboptimal and optimal doses were chosen based on previous studies (Le [START_REF] Hir | AAV Genome Loss From Dystrophic Mouse Muscles During AAV-U7 snRNA-mediated Exon-skipping Therapy[END_REF], and rAAV2/9-mSEAP vectors were injected in Tibialis anterior (TA) muscles.

About 50 µl of blood was weekly withdrawn from the tail vein during 4 weeks (Figure 23a). After sacrifice, cryosections of TA were stained for mSEAP product detection.

Microscopic observations show that whatever the purification process used, rAAV vectors transduced the muscle with good efficiency. Nevertheless, we observed that TC-derived vectors gave better spreading of mSEAP-positive fibers and higher staining intensity around the site of injection than CP-and SN-derived vectors (Figure 23b). To confirm the histological data, a quantitative assessment was performed by measuring the kinetics of mSEAP concentration in blood during the 4 weeks postinjection (Figure 23c and Supplementary Table 4). Surprisingly, TC-derived rAAV vectors performed better in vivo mSEAP secretion after intramuscular delivery than those issued from other classical methods. These in vivo results consolidate the in vitro observations and confirm that our new purification method gives more functional rAAV vectors, probably due to better conservation of the vector quality and integrity. 4 and Supplementary Figure 24b for more detailed table and pictures from TA muscles injected at higher rAAV doses.

Discussion

rAAV downstream process is continuously evolving. Such developments allow better yields and purity, increasing the interest of these vectors for gene therapies.

Here, we show that such improvements can be obtained following fast and ergonomic purification process. Instead of focussing our procedures on producer cells (CP) or on the supernatant (SN) separately, we decided to recover the vectors from the whole cell culture by using simple detergent-mediated cell lysis at the end of the production stage. Triton X-100 is a commonly used detergent in molecular and cell biology laboratories. It is a nonionic surfactant that has a hydrophilic polyethylene oxide and an aromatic hydrophobic group (Figure 20b). Its high solubility in large panel of aqueous solutions makes it the compound of choice for permeabilization of mammalian cell membranes, even in their classical culture environment. Moreover, its nonionic property is widely used to solubilize proteins while maintaining their native structure and functionality, contrary to some denaturant ionic detergents, such as SDS. One hour of 0.5% Triton X-100 treatment is sufficient for the release of intracellular rAAV from cells and added up to vector particles already present in the supernatant (originating from exocytosis or "natural" cell lysis). Our results show that Triton X-100 treatment of TC allows higher recovery of particles if compared to CP or SN purification methods. In most cases (productions #1 and #2, Supplementary Table 2) total amount of vg issued from TC procedure is equivalent to the sum of vg from CP and SN methods. Furthermore, our previous experimental observations and RNA/DNA ratio quantifications (averaged between 6 and 10) lead us to consider RNA contamination as principal cause of supernatant viscosity. This criterion, combined with the crucial role of the isopycnic iodixanol gradient as physical barrier for genomic and plasmid DNA molecules, lead us to eliminate the cost-prohibitive DNAse from the process without altering the quality of the final product. This approach, combined with reduced process duration, highly purified product, more ergonomic and less expensive procedure, constitutes a real improvement in rAAV purification method.

Release of intracellular content following cell lysis could be detrimental for the expected final purity of our viral vectors. Nevertheless, optimization of downstream technologies in this last decade has provided numerous robust and efficient equipments and disposables able to face many hurdles on purification processes.

Among them, the dead-end filtration capsules, used consecutively to cell lysis, offer wide range of products with different kind of membranes (polysulfone, polyethersulfone, glass fibers, etc.) and large panel of pore sizes or molecular weight cut-off, able to significantly increase the efficiency of the clarification step in terms of debris removal and viscosity reduction (by nucleic acids capture). Otherwise, the concentration step is becoming a technology hub in the process since volume reduction simplifies the subsequent procedures, drastically diminishes the duration of the total process, and removes more contaminants. Nowadays, TFF systems propose also a wide range of fibers (hollow fibers or cartridges) with various membrane types and surfaces, and a variety of molecular weight cutoff. Moreover, complete integrated automatic systems for research or pilot scales are now commercially available, allowing perfect control of pressures, concentration levels, and flow rates. In our approach, TFF system remains central tool to treat volumes issued from cell culture vessels such as Hyperflasks, which generate up to 1 l of total cell culture suspension. By judiciously combining molecular weight cutoff, membrane surface and material assembly, TFF approach is currently able to reach 2-to 80-fold concentration levels. Such technological performances offer possibilities to easily implement rapid and low cost ultracentrifugation approaches into the process. These methods, especially those using iodixanol isopycnic density gradients [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF] remain advantageously simple not only to remove nucleic acids from fractions containing vectors but also to separate empty and full rAAV particles from initial bulk. In our hands, 1L of clarified lysate is 30-fold concentrated before to be run through only two equilibrated ultracentrifuge tubes. According to these concentration capacities, the classical Ti70 angle-fixed ultracentrifuge rotor (Beckman Coulter, France), having eight-tube compartments, could for instance be used to treat up to 10L of original crude lysate in one unique run of less than 2 hours. These technical characteristics offer the opportunity to use the most diffused purification approach for scalable processes as those employing baculovirus/insect cells systems [START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF], competing with some well-described chromatographic methods [START_REF] Okada | Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes[END_REF][START_REF] Qu | Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography[END_REF][START_REF] Urabe | Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression[END_REF] that require expensive equipment and more technical knowledge and practice than ultracentrifugation use.

The most interesting and surprising effect of the Triton X-100 treatment on producer cell lines is the generation of rAAV vectors showing better in vitro and in vivo efficiency than their homologous particles issue from classical purification methods (cell pellet or supernatant). Several hypotheses could explain this efficiency.

The CP clarification process is somewhat drastic, especially during the cell lysis step.

The physical cell disruption method, using several cycles of freezing/thawing can alter integrity of viral capsids without completely destroying the genome-full particles (that can still be quantified by quantitative PCR). In another hand, SN-derived vectors could be released in supernatant long before the end of the production phase. The progressive acidification of the culture medium during upstream process, resulting from the cell metabolism, in addition to the presence of proteases originating from producer cell lysis both provide an unfavorable environment for released rAAV particles and can lead to structural alteration of the SN-derived vectors. In vivo analyses (Figure 23c) were performed following intramuscular delivery of rAAV-mSEAP. Murine-secreted alkaline phosphatase was previously described as excellent reporter gene due to its non-immunogenic property that allows non-invasive monitoring of gene transfer [START_REF] Bartoli | Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies[END_REF][START_REF] Maelandsmo | Use of a murine secreted alkaline phosphatase as a nonimmunogenic reporter gene in mice: Murine Alkaline Phosphatase as a Reporter Gene[END_REF]. In this study, we clearly show a better mSEAP product expression in blood of mice injected with TC-derived rAAV compared to those injected with SN-or CP-derived vectors.

Following our argumentation, we can suppose that the detergent treatment avoids the use of techniques that alter the physical integrity of viral particles and thus enrich the proportion of fully functional rAAV.

These last years, continuous development of upstream and downstream processes has led to successful improvements of several quantitative and qualitative characteristics of rAAV as the yields, purity, and scalability. These ameliorations were also combined with reduction of process durations and reduction of the costs, two important criteria in industrial technologies dedicated to clinical aiming.

Materials and Methods

Upstream process

A multilayer Hyperflask (Corning, The Netherlands), with a total growth area of 1,720 cm 2 , is seeded following manufacturer instructions with 35 × 10 6 HEK293T cells (corresponding to 2.0 × 10 4 cells per cm 2 ) in total volume of 560 ml of 1× Dulbecco's modified Eagle medium supplemented with Glutamax, 4. Transfected cells are incubated for 72 hours at 37 °C under 5% CO2.

Silver staining of SDS-PAGE

Purity of recombinant AAV vectors is assessed by loading 1 × 1010 vg on SDS-PAGE (Life Technologies) run under reducing conditions. Proteins and nucleic acid content is revealed by using Silver Staining Plus protocol (Bio-Rad Laboratories, France) following manufacturer instructions.

In vitro assay

In vitro assays are performed in 24-well tissue culture plates having 2 cm2 of growth area per well. 2 × 10 5 HEK293T cells per well are seeded in total volume of 300 µl of complete medium (described before). After 24 hours, an average of the number of cells per well is determined by trypsinization and automatic cell counting. Vector transduction is performed at multiplicity of infection 105. Fourty-eight hours later, the medium is collected and conserved at -20 °C until use. The cell monolayers are washed once with 1× PBS and fixed with 0.5% glutaraldehyde solution for 15 minutes at room temperature before three washing cycles of 15 minutes with 1× PBS. To inactivate endogenous alkaline phosphatase activity, cells are covered with 500 µl of PBS and plates are sealed and immersed in water bath heated at 65 °C for 30 minutes.

After two PBS washings, 5-Bromo-4-Chloro-3 Indolyphosphate/Nitro Blue Tetrazolium solution (Sigma-Aldrich) is added in each well and plates are incubated at 37 °C for 30 minutes to several hours until occurrence of significant visual colored staining, compared to non-transduced cells. The mSEAP quantification in collected medium is performing with the Phospha-Light SEAP Reporter Gene Assay System (Life Technology) according to manufacturer instructions. Briefly, medium is first 10fold diluted in a dilution buffer provided with the Phosphalight kit before endogenous alkaline phosphatase inactivation at 65 °C for 30 minutes. After sixfold serial dilutions in assay and reaction buffers containing chemiluminescent substrates and enhancers, microplates are placed in luminometer for luminescence detection.

Raw data were analyzed through simple spreadsheet and quantified by using standard curve previously prepared by serially diluting stock enzyme.

In vivo assay

All studies on mice were conducted in strict accordance with the institutional guidelines for animal research transposed from the European directive 2010/63/EU. Project authorization, including all the procedures, was submitted to Ministry of Higher Education and Research on March 31, 2014. All injections were performed after inhalational anesthesia using isoflurane, and all efforts were made to minimize animal number, procedure lengths, and suffering.

Ten-week-old C57BL/10 mice (three animals per group) were injected intramuscularly with 50 µl of AAV2/9-mSEAP vectors issued from CP, SN, or TC purification process. A total of 5.0 × 10 10 vg (high dose) and 1.0 × 10 10 vg (suboptimal low dose) were injected in left and right TA muscles of each mouse respectively. Fifty microliters of blood were withdrawn every week for 1 month, and serum was isolated by blood centrifugation at 10,000 × g for 5 minutes at 4 °C. Mice were sacrificed 1 month post-injection by cervical elongation and TA muscles are harvested.

TA muscles are immediately frozen in liquid nitrogen-cooled isopentan solution and cryopreserved at -80 °C. Serial 10-µm cryosections were made and examined for alkaline phosphatase enzymatic activity. Histological slides are first fixed with 0.5% glutaraldehyde solution for 15 minutes at room temperature before three cycles of 15 minutes washing with 1× PBS. To inactivate endogenous alkaline phosphatase activity, slides are incubated at 65 °C for 30 minutes in PBS, followed by two PBS washings. 5-Bromo-4-Chloro-3 Indolyphosphate/Nitro Blue Tetrazolium solution (Sigma-Aldrich) is added to cryosections and incubated at 37 °C for 30 minutes to several hours until occurrence of significant visual colored staining, compared to slides of non-injected muscles.

Quantification of mSEAP from mice serum is performed as described for culture medium. The only difference is the initial serum is fivefold more diluted than used with supernatant from in vitro experiments.

Statistical analyses

Graphical representations are presented as mean ± standard error, and a twotailed Student's t-test is used to determine significant differences in total viral genome recovery between TC-and SN-derived vectors (Figure 21b) or for in vitro mSEAP product quantification between TC-and SN-derived vectors (Figure 22b). P < 0.05 (represented by * in Figure 21b) and P < 0.01 (represented as ** in Figure 22b) are considered as significant and highly significant respectively.

II. Article 2: Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

Authors: Buclez PO, Dias-Florencio G, Relizani K, Beley C, Garcia L, Benchaouir R.

Introduction

Recombinant adeno-associated viruses (rAAV) are powerful tools for gene transfer and are currently used in research, preclinical, and clinical developments.

These vectors display significant advantages: they are well tolerated after in vivo delivery, allow long-lasting gene expression, and numerous serotypes with specific tropisms are also available [START_REF] Balakrishnan | Basic biology of adeno-associated virus (AAV) vectors used in gene therapy[END_REF]. The first marketing approval for rAAV gene therapy has recently been delivered for the treatment of familial lipoprotein lipase deficiency [START_REF] Gaudet | Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial[END_REF][START_REF] Salmon | Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera ® )[END_REF]. It is likely that similar therapies will soon be approved for other pathologies [START_REF] Kotterman | Engineering adeno-associated viruses for clinical gene therapy[END_REF], calling for vector production improvements. Scale-up processes are rapidly evolving even though needs are different according to the type of disease to treat. Indeed, the rescue of Leber's Congenital Amaurosis defect by rAAV vectors only requires the injection of less than one milliliter of product underneath the retina [START_REF] Bainbridge | Effect of Gene Therapy on Visual Function in Leber's Congenital Amaurosis[END_REF][START_REF] Hauswirth | Treatment of Leber Congenital Amaurosis Due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results of a Phase I Trial[END_REF][START_REF] Maguire | Safety and Efficacy of Gene Transfer for Leber's Congenital Amaurosis[END_REF], while treatment of Duchenne muscular dystrophy is more challenging and would obviously solicit much higher volumes of viral vectors to target the whole body musculature [START_REF] Yue | A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs[END_REF]. Different upstream processes exist to meet the quantitative requirements in viral vectors. Small-scale productions can be performed through the classical tri-transfection method in adherent human embryonic kidney 293 (HEK293) cell line [START_REF] Grimm | Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors[END_REF][START_REF] Salvetti | Factors Influencing Recombinant Adeno-Associated Virus Production[END_REF][START_REF] Xiao | Production of high-titer recombinant adenoassociated virus vectors in the absence of helper adenovirus[END_REF]. For large-scale productions, methods using baculovirus expression vector system were successfully implemented to produce rAAVs by insect cell lines grown in suspension (Mietzsch et al., 2014;[START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF]. Alternative methods using specific HEK293 cells strains able to grow in suspension were also described [START_REF] Chahal | Production of adenoassociated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery[END_REF]. Other studies have shown the potential for producing rAAV vectors by stable cell lines [START_REF] Martin | Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production[END_REF] or the use of Herpes simplex virus for manufacturing productions [START_REF] Ye | Herpes Simplex Virus Clearance During Purification of a Recombinant Adeno-Associated Virus Serotype 1 Vector[END_REF]. On another hand, downstream processes propose a large panel of methods.

Among them, chromatographic approaches are generally preferred when the levels of production become compatible with industrial scales. The most efficient ones use either ion exchange [START_REF] Okada | Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes[END_REF][START_REF] Qu | Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography[END_REF][START_REF] Urabe | Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression[END_REF] or affinity methods [START_REF] Anderson | A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction[END_REF][START_REF] Auricchio | Isolation of Highly Infectious and Pure Adeno-Associated Virus Type 2 Vectors with a Single-Step Gravity-Flow Column[END_REF][START_REF] Koerber | Engineering Adeno-Associated Virus for One-Step Purification via Immobilized Metal Affinity Chromatography[END_REF]. Nevertheless, these techniques have the drawback of being relatively serotypespecific and require the implementation of other purification techniques, thus accentuating yield losses. Besides that, the costs of specific resins used to pack columns are quite expensive and detrimental during the launch of a clinical project. Finally, despite some described analysis methods [START_REF] Lock | Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography[END_REF], these approaches currently remain ineffective for the separation of empty particles from full ones. Thus, implementation of alternative purification approaches for large-scale productions should be proposed to circumvent the qualitative, quantitative, and financial constraints mentioned above. One option is to simply use the physical properties of the viral particles to provide processes based on their density or sedimentation rate. Indeed, ultracentrifugation systems based on density gradients are successfully used for rAAV purification at research scale; iodixanol-based gradients appeared more suited for AAV vectors than the conventional cesium chloride (CsCl)-based gradients [START_REF] Hermens | Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System[END_REF][START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. In this context, we recently showed that a simplified AAV purification process from HEK293T producer cells, using an isopycnic iodixanol gradient, led to high vector recovery and an unexpected improvement of in vitro and in vivo functionalities compared to classical described protocols [START_REF] Dias Florencio | Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors[END_REF]. The ultracentrifugation process is generally considered too difficult or even impossible to be implemented in the handling of largescale productions. The relatively low volume of supernatant that can be treated by a single run of ultracentrifuge explains in large part the reluctance of users to choose this technology when processing large volumes. Nevertheless, equipments designed to highly concentrate big volumes of biological solutions are now available, making it possible to consider ultracentrifugation as a feasible method for the final purification step. Indeed, this approach has been successfully applied to purify rAAV from several liters of crude lysates, although the process extended over 1 week [START_REF] Lock | Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale[END_REF].

Here, we show that implementation of an optimized isopycnic gradient construction led the process to be advantageously used for treating large initial volumes of vector as well as reducing the entire purification step to one working day.

Compared to purification based on conventional iodixanol gradients [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF], our method equally preserved high final product quality and the rAAV particles remained functional as shown by in vitro and in vivo transduction assays.

Results

Optimized isopycnic gradient method for efficient rAAV purification

The rAAV upstream process was performed by using the baculovirus expression vector system (Supplementary Figure 30). We used different recombinant baculoviruses, one harboring the codon-optimized rep2 and cap9 genes under potent baculovirus promoters [START_REF] Smith | A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells[END_REF][START_REF] Urabe | Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression[END_REF] and the others harboring either the green fluorescent protein (GFP) or the mouse secreted alkaline phosphatase (mSEAP) reporter genes under a cytomegalovirus (CMV) promoter (Supplementary Figures 30 and33). After generating the baculovirus-infected insect cells (BIIC) [START_REF] Cecchini | Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02-to 200-Liter Cultures[END_REF][START_REF] Wasilko | The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus[END_REF], rAAV productions were initiated by cocultivation of two BIICs, including BIIC-Rep2opt/Cap9opt with either BIIC-GFP or BIIC-mSEAP, with native Sf9 insect cells in a ratio of 1 to 104 v/v respectively. Of note, all cell cultures were performed in serum-free medium. Under these conditions, the upstream process was stopped 72 hours later, after which the downstream process was initiated. Figure 27a is a schematic representation of the downstream process. The latter is described in further detail in Supplementary Table 5. The first clarification step started with the addition of 0.5% Triton X-100 detergent in the culture to induce efficient cell lysis without altering the viral particles as we previously described (Dias minutes with slightly modified conventional method) and the length of total purification process by 3× factor. The new approach allowed the collection of lower volume of sample after ultracentrifugation, leading to supplemental time reduction of the diafiltration phase. Below the scheme are shown the time scale for each of the downstream process steps. The total time gain between the conventional and novel processes is shown at the bottom right of the figure. Further details on the downstream process are described in material and methods and Supplementary Table 5.

The most significant downstream process improvement was achieved with the implementation of a new iodixanol isopycnic density gradient. A more conventional purification method, derived from the original work of Zolotukhin et al. [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF], was compared to our optimized process (Figure 27 and Supplementary Table 5). As shown in Figure 27, two major types of changes were made to this central process step. The first one was related to the construction of the iodixanol gradient; while the conventional method used four layers of iodixanol at different densities, our new protocol only used three layers since the 15% density layer was removed. The volumes of each remaining layer were also substantially reduced (3 ml of 25%, 4 ml of 40% and 2 ml of 60% against 6 ml of 25%, 5 ml of 40%, and 3 ml of 60% for the new and classical method, respectively). These modifications allowed the filling of nearly twice the volume of viral suspension (30 ml) per ultracentrifugation tube then that previously described [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. In addition to reducing the number of tubes by half, these changes also reduced the filling process time (from 10 to 5 minutes per tube for classical and new method respectively). Furthermore, the new purification method allowed the recovery of the viral vectors after 20 minutes of ultracentrifugation (at 70,000 rpm) against 80 minutes using our previous slightly modified conventional method [START_REF] Dias Florencio | Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors[END_REF]. Following this new protocol, one round of ultracentrifugation instead of two was performed to treat equivalent volumes of production. Moreover, only 4.5 ml of ultracentrifuged product instead of 6 ml was collected, allowing a twofold reduction in the number of diafiltration systems used in the following step. This feature reduced the length of the last purification step as described in Figure 27 and Supplementary Table 5. Finally, unlike the conventional method (lasting 9.5 hours), our optimized method used to treat pilot productions of more than 5 l (> 1 × 10 14 viral vectors) allowed to reduce the length of the entire downstream process to a single working day (6 hours).

The new process did not alter productivity, quality, and in vitro efficacy of rAAV vectors

The implementation of a new purification method raised the question of its effectiveness in giving equal or even higher vector yields than those obtained using the conventional method. This question was first analyzed through quantitative titration of rAAV. As shown in Figure 28a and Supplementary Table 6, no significant difference in titers was found between the two approaches (7.8 × 10 12 and 5.2 × 10 12 total vg purified by classical and new method respectively, recovered from three distinct 100 ml productions).

Moreover, we analyzed the quality of viral vectors by using 5 × 10 10 vg from each purification process by running a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that was thereafter treated for silver staining (Figure 28b). We found no difference in quality between the vectors obtained from the two purification methods. Indeed, in both cases, the level of purity was satisfactory since the conventional viral capsid protein ratio was maintained in both situations (ratio of 1/1/10 for VP1/VP2/VP3 respectively). Of note, on silver-stained SDS-PAGE, we detected a few thin bands which most likely were the result of degraded VPs by baculoviral proteases as previously described [START_REF] Hitchman | Genetic modification of a baculovirus vector for increased expression in insect cells[END_REF][START_REF] Kaba | Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins[END_REF]. Furthermore, we compared rAAV-GFP vectors through an in vitro transduction assay, followed by flow cytometry analysis for a precise quantification of GFP-positive cells. HEK293T cells were transduced with rAAV-GFP recovered from the conventional or new purification methods at different multiplicity of infection.

Seventy-two hours post-transduction, cells were visualized by fluorescent microscopy before flow cytometry quantification (Figure 28c, d and Supplementary Figure 31). Independent of the multiplicity of infection used, no significant difference in in vitro transduction efficacy was observed between vectors collected from the two methods of purification. Moreover, no cytotoxic effect was observed following microscopy analyses (Figure 28d). We concluded that the new purification process allowed recovering vectors of high quality without any alteration in yields and in vitro transduction capacity compared to the rAAV purified by conventional downstream process. rAAV products by silver staining of 5.10 10 vg loaded on SDS-PAGE. The new process lead to same purity levels as the classical method. Capsid proteins VP1, VP2, and VP3 were identified, showing same relative ratio (about 1/1/10 for VP1/VP2/VP3 respectively) for the two processes. (c) Flow cytometry quantification of 72 hours post-in vitro transduction of HEK293T cells at multiplicity of infection (MOI) of 10 3 showed no significant difference in percentage of GFP-positive cells between vectors recovered from the conventional or the new purification method. (d) Fluorescent microscopy of the HEK293T cells 72 hours post-rAAV transduction confirmed the equal efficacy of the two processes to generate functional rAAV without altering cell survival or inducing cell death as observed by homogeneous cell monolayer in both cases (refer also to Supplementary Figure 31 for more results). (e) Graphical representation of the quantity of total vg extracted from the first 6 ml fractions collected from suspensions processed with new method (n = 3). A peak was observed inside the fraction 3, corresponding to the first milliliter of the 40% iodixanol phase. After a reduction of viral genomes in fraction 4, we observed a slight increase in fractions 5 and 6. The vertical dotted line delineates the collected fractions from the rest of uncollected ultracentrifuged suspension. (f) Silver staining of a SDS-PAGE in which identical volumes of each iodixanol fractions were loaded. The relative intensity of the rAAV capsid proteins (VP1, VP2, and VP3) appeared to change similarly to the amount of viral genomes presented in (e). Delimitation between collected and uncollected fractions is represented in the bottom of the gel by a vertical dotted line. (g) Fluorescence-activated cell sorting (FACS) analysis of GFP-positive HEK293T cells transduced at MOI of 10 2 by rAAV collected from the first 4.5 ml or the next 1.5 ml fraction produced from the new method (n = 3). Significant difference in percentage of GFPpositive cells was observed between the two fractions as shown by *(P = 0.03), indicating the presence of less efficient or defective vectors in the fractions located above the first 4.5 ml. An example of FACS gates is shown in the right part.

Iodixanol gradient fractionation revealed the distribution of functional and altered rAAV particles

To better characterize the distribution of vector particles inside the new iodixanol gradient set up (Figure 27), we collected milliliter fractions from the bottom of the ultracentrifuged tubes, followed by analysis of the viral genome content by quantitative polymerase chain reaction (Q-PCR). As shown in Figure 28e, the graphical distribution of vg gave a Gaussian-like curve with a peak observed in fraction 3 containing collected volumes ranging from 2 to 3 ml. According to the new set up of iodixanol gradient (Figure 27), fraction 3 could correspond to the boundary between the 60% and the 40% phases, confirming the cushion property of the 60% layer, and the role of the 40% layer as an equilibrium phase for the full viral particles [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. Figure 28e also shows a rise in the level of vg from fraction 5 (volumes ranging from 4 to 5 ml). Since only the first 4.5 ml are collected from our new purification method, we wondered about the nature of these "upper layers viral particles". Of note, we decided to collect the first 4.5 ml rather than the first 6 ml, as carried out with the conventional method, since we observed a significant decrease in the quality of purified products when we collected far from fraction 4 (data not shown). After running 10 µl of each milliliter collected fractions on SDS-PAGE, the gel was subjected to silver staining. As depicted in Figure 28f, the VP band intensities showed a great quantitative correlation with the titration results shown in Figure 28e.

Moreover, these results clearly confirmed the deterioration of purity, visually indicated by dark smears, as we collected closer to and mostly after fraction 6.

According to the new gradient construction (Figure 27), the upper boundary of fraction 6 should correspond to the limit between the 40 and the 25% layers, explaining the possible new cushion property of this border able to stop many potential contaminants.

Finally, we compared the in vitro transduction efficacy of the first 4.5 ml with the following 1.5 ml to determine whether the particles detected by Q-PCR (Figure 28e) and silver staining (Figure 28f) in this last uncollected fraction were still fully functional. A multiplicity of infection of 10 2 was used on HEK293T cells and the percentage of GFP-positive cells was quantified by flow cytometry analysis 72 hours post-transduction (Figure 28g). A significantly higher transduction efficacy was observed when using rAAV particles collected from the first 4.5 ml fraction compared to the following 1.5 ml fraction of the iodixanol gradient (P = 0.03). This result showed that the "upper layer particles", as illustrated in Figure 28e, probably contained a large majority of defective interfering particles. The lower density of these defective vectors could suggest the presence of incomplete viral genomes. Of note, it was technically difficult to concentrate fractions beyond the sixth phase due to the high concentration of contaminants causing filter clogging during the buffer exchange procedure. Due to the inability to obtain volumes as concentrated as the previous phases, we did not explore further the content in particles of the upper iodixanol gradient fractions.

Nevertheless, we hypothesized that empty rAAV particles might be retained in the 25% phase of the gradient. More extensive subsequent studies will be performed to better characterize the full to empty particle ratio obtained following our process.

The new process was applicable to pilot-scale productions

To determine whether this novel process could be applied to larger scales, we processed up to 400 ml of productions using Erlenmeyer flasks, and 5L using bioreactor system. The quantification of the total number of viral genomes is shown in Figure 29a and gave a linear rise with 5.1 × 10 12 , 3.1 × 10 13 , and 2.3 × 10 14 total vg purified from three independent processes of 100 ml, 400 ml, and 5L productions, respectively. Supplementary Table 5 online gives supplementary titration details, showing, for example, a relative constant yield (vg/cell) whatever the volume of production. We also evaluated the quality of the vectors collected from the 5L process by silver staining of SDS-PAGE (Figure 29b). A high-quality profile was obtained, also respecting the conventional ratio of the natural respective levels of VP1, VP2, and VP3

(1/1/10). Together, these quantitative and qualitative results provided the proof of principle that the optimized process met the requirements for scalable levels of viral vector productions.

In addition, taking into account the previous in vitro transduction results between the conventional and the new methodological approaches (Figure 28c,d), we considered that only an in vivo transduction assay could truly answer the question of the functionality of rAAV vectors. We focused our investigations on serotype 9 of the AAV, which is well documented for its tropism for muscle tissues [START_REF] Fang | Comparison of Adeno-Associated Virus Serotypes and Delivery Methods for Cardiac Gene Transfer[END_REF][START_REF] Katwal | Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery[END_REF][START_REF] Kornegay | Widespread Muscle Expression of an AAV9 Human Mini-dystrophin Vector After Intravenous Injection in Neonatal Dystrophin-deficient Dogs[END_REF][START_REF] Schinkel | Long-Term Preservation of Cardiac Structure and Function After Adeno-Associated Virus Serotype 9-Mediated Microdystrophin Gene Transfer in mdx Mice[END_REF]. We decided to replace the GFP with the mSEAP reporter gene to allow a non-invasive in vivo monitoring as previously described [START_REF] Bartoli | Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies[END_REF]. A group of three C57BL/10 adult mice was first intramuscularly injected in Tibialis anterior (TA) muscle with 2 × 10 12 vg of rAAV2/9-mSEAP vectors. mSEAP protein was weekly monitored in blood for 4 weeks. Figure 29c shows the gradual rise of mSEAP levels in blood over time, compared to phosphate-buffered saline (PBS)-injected mice. One month after vector injection, mice were sacrificed and injected muscles were stained to reveal the levels of mSEAP produced by muscle fibers. Saturating mSEAP signal was observed all over the injected TA (Figure 29d), confirming the great functionality of the rAAV vectors in an in vivo environment. To go one step further, we took advantage of high titers obtained from purifications of the 5 L productions to perform whole body treatments by systemic administration of the vectors in order to assess the tissue tropism and global biodistribution of our vectors. Retro-orbital injections of a group of three C57BL/10 adult mice were performed. We collected blood 7 days later and tissues 1 month post injection (Supplementary Figure 32a). As shown in Supplementary Figure 32b, quantification of the levels of mSEAP in blood 7 days post injection was compared to the one analysed in intramuscularly injected mice, and was found to be more than 15-fold higher with 1,600.3 and 108.3 ng of mSEAP per ml of blood between systemic and intramuscular-injected mice respectively. In parallel, different tissue cryosections were stained for mSEAP expression. TA, Triceps brachialis, hearts, diaphragms, kidneys, and livers were all strongly positive for mSEAP compared to the PBS-injected control mice (Figure 29e and Supplementary Figure 32c), demonstrating the ability of our rAAV2/9 vectors to be largely biodistributed all over the body. The 100 and 400 ml suspensions were produced in Erlenmeyer flasks whereas the 5l were done in a bioreactor system. Supplementary details on titers and yields are available in Supplementary Table 6. (b) The quality of the final rAAV product recovered from 5l production was determined by silver staining of 5 × 10 10 vg loaded on SDS-PAGE. The same high-quality vectors than previously described for research-scale productions were obtained (see Figure 28b). The VP ratio was also maintained.

Discussion

Here we show that it is possible, by implementation of simple procedures, to propose a new, rapid, low-cost, and robust rAAV process able to meet the demands of many laboratories in term of quantity and quality of vectors. The use of baculovirus/insect cell system has made a decisive turn toward the industrial mode of production, a required milestone for the use of innovative therapies based on gene transfer to future patients with cancer, genetic, or neurodegenerative diseases. The baculovirus/insect cell system confers ultimately the possibility of scaling up the levels of production much more than can be provided by processes using HEK293T adherent cells. This flexibility in volumes of production is an advantageous characteristic for institutions practicing research and development and interested in advancing their therapeutic projects from the laboratory to the clinic. It is important to notice that the baculovirus expression vector system is no longer the only proposed production system capable of scaling up the levels of production. Competing upstream process approaches have emerged such as those using derived HEK293 cells able to grow in suspension [START_REF] Chahal | Production of adenoassociated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery[END_REF] or the use of stable cell lines or Herpex simplex virus [START_REF] Martin | Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production[END_REF][START_REF] Ye | Herpes Simplex Virus Clearance During Purification of a Recombinant Adeno-Associated Virus Serotype 1 Vector[END_REF]. These interesting systems, using transfection protocols, remain nevertheless highly dependent on large quantities and good quality of plasmids required for the production of viral vectors, characteristics particularly important in view of good manufacturing processes grade productions for the delivery of clinical batches. Alternatively, the use of BIIC as raw material necessary to start rAAV production in insect cell cultures appears as another turning point in the quest for baculovirus cryo-conservation, but also for simplification and significant reduced costs of the upstream process [START_REF] Cecchini | Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02-to 200-Liter Cultures[END_REF]. The use of infectious recombinant baculoviruses allows subsequent amplification of vector production that is finally extended to the whole initial insect cell culture 72 hours post-BIIC inoculation. For example, by cryo-preserving 200 ml of BIIC per new recombinant baculovirus, constituting a master cell bank (Supplementary Figure 30), we could theoretically process about 2,000 l of rAAV production. Obviously, the initial volumes of produced BIIC could be amplified well beyond the previous example.

The advantages conferred by the rapid scaling up of production levels necessarily raise the question of the subsequent purification steps. Here, we show that two important phases of the downstream process allow significant improvements in terms of duration and consequently sample volumes able to be processed. The first important point concerns the concentration process by tangential flow filtration (TFF).

There is currently a large number of concentrating systems available from different manufacturers. Tangential ultrafiltration hollow fibers or cassettes exhibit a wide range of products differentiated by their cut-off, the nature of the membranes, and the filter surfaces. By rigorously establishing all of these parameters on small-scale productions, as we previously performed, it becomes possible to easily define their equivalents for larger volumes. Moreover, the availability of automated TFF systems, able to control both the transmembrane pressure as well as the levels of concentration factors provide an ergonomic and secure environment to the manipulator, and facilitate even more the speed of the process. These developments now allow us to ensure a relative homogeneity in the TFF duration between small and large volumes for identical concentration factors. Indeed, by defining, for instance, a constant filtrate flux rate of 15 l/m 2 /hour, we can easily calculate the required filter surfaces related to the original sample volumes to be treated. Here, we confirm that this process can be performed with a difference of only 1 hour of TFF treatment between our research and pilot-scale productions. Indeed, as described later, 25L of sample have required less than 3 hours to be 30× concentrated (higher concentration factor than used in this work).

On another hand, the real novelty presented here concerns the important optimization made to the set up and processing of the iodixanol density gradient. The majority of publications describing purification of AAV vectors by discontinuous iodixanol gradients refer to the initial work of Zolotukhin et al [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. As presented in this work, the published gradient set up was originally done by successive deposition of four layers of iodixanol solutions at various concentrations (15, 25, 40, and 60%) over which the vector suspension is deposited. In the original article, the authors have used a first 9 ml phase of 15% iodixanol at 1 M NaCl. Addition of NaCl was justified as necessary to destabilize ionic interactions between macromolecules and AAV particles. This necessity was implemented in the process because the iodixanol gradient was used immediately after cell lysis, followed by lowspeed centrifugation to simply remove the resulting large cell debris. In our protocol, clarification step using detergent and nucleases followed by tangential ultrafiltration drastically reduced the macromolecular complexes in the viral suspension before its deposit on iodixanol gradient. Indeed, in these experimental conditions, the viscosity of our concentrated solution was relatively low. The first reason is the consequence of nucleases treatment performed during cell lysis. The other point concerns the use of serum-free medium instead of serum-supplemented media used for productions performed in HEK293T cell cultures. Altogether, these observations led us to deeply change the construction of iodixanol gradients with the objective of both increasing the initial amount of viral suspension that can be processed per run and decreasing the time of ultracentrifugation. Our results show that the classical 15% phase of the iodixanol gradient (at 1M NaCl) is clearly dispensable, allowing its replacement by equivalent volume of vector suspension. The adjustment of the volumes of the other phases to 2 ml, 4 ml and 3 ml instead of 5, 5, and 6 ml for the 60, 40, and 25% phases, respectively allow us to fill 30 ml of concentrated vectors per ultracentrifuge tubes (39 ml maximum volume capacity) instead of 15 ml as specified in the original publication. The twofold increase of the volumes per tube coupled to the upstream TFF concentration factor significantly increased the purification capacity of the iodixanol gradient approach. Moreover, after several empirical assays, we were surprised to observe that no significant difference in titers was found between suspensions ultracentrifuged for 80 or 20 minutes, a fall of titers becoming significant for shorter periods (data not shown). Knowing that a Ti70 rotor type supports up to eight tubes of ultracentrifugation, it is possible to treat up to 240 ml of viral suspension in just 20 minutes. If this volume of viral suspension derived from a usual 30 to 40 times concentrated solution, it then turns out that a single 20 minutes run of ultracentrifugation is able to process a starting raw production of approximately 10L.

Considering the fact that less than 30 minutes are now sufficient to prepare series of 8 density-gradient tubes, it becomes easy to imagine the possibility offered by this new approach to perform nearly a dozen runs per working day and per single manipulator without many technical difficulties. In an industrial context, one can imagine to advantageously implement series of ultracentrifuge systems to easily multiply the level of purification capacity offered by our new iodixanol gradient approach.

Therefore, we believe that such a process, considered originally as restricted solely to research scales due to the limitation imposed by the volumes able to be treated by ultracentrifugation, can now be considered for the purification of pilot or even industrial-scale productions. Of note, the following diafiltration step, using in this work centrifugal filtration units as reformulation systems, can easily be replaced by TFF systems to be more compatible with industrial requirements. Finally, the costs generated by our downstream process can be considered negligible compared to competing described processes as previously discussed in the introduction.

These promising process developments obviously pushed us to compare and verify the yields, quality, but also the efficiency of AAV vectors generated by our new technological approach. Our quantitative results show a good linearity between research and pilot-scale productions made in Erlenmeyer flasks and bioreactor respectively. Moreover, the codon optimization of the rep and the cap genes was an important prerequisite for optimal expression of the resulting proteins in an insect cell environment [START_REF] Smith | A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells[END_REF][START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF]. Our results clearly confirm the well-conserved quality of the viral vectors by using our new purification approach, and no significant difference in the in vitro transduction efficacy was observed between vectors recovered from the conventional and the optimized process.

Nevertheless, a subsequent analysis of the full to empty particle ratio will be necessary to complete the quality control of our new AAV process.

In addition, newly processed rAAV vectors produced at scale (5L) showed high intramuscular efficacy. The results from intravenous administration also demonstrated the capacity of these vectors to spread all over the body through transduction of muscular and non-muscular tissues. Altogether, these observations confirm once again that the optimized process does not affect the quality and functionality of AAV vectors.

One could point out the fact that in this study we did not exceed volumes of production higher than 5L. Our first response is that technology of production by bioreactors automated systems is strictly controlled and designed to allow a gradual transition to the large volumes, thus keeping linearity between production levels. The same linearity characteristic is found with the tools and technologies available for the downstream processes. Moreover, as briefly discussed above, we recently processed 25L productions for treatment of Golden Retriever muscular dystrophic dogs, a canonical animal model of Duchenne muscular dystrophy [START_REF] Vulin | Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping[END_REF]. The titers obtained by the implementation of our new downstream process exceeded 10 15 vg, opening the door to whole body treatments on adult dogs for preclinical studies. The technological details and results of this project will be the subject of a future communication. Nevertheless, our results show that this optimized process is compatible with the scaling up of the vector productions towards larger volumes, maintaining high titers, short process duration, and low costs.

Material and methods

Recombinant baculovirus and BIIC production processes

Codon-optimized Rep78/52 gene (from AAV2 serotype) was generated as previously described [START_REF] Smith | A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells[END_REF]. The codon-optimized Cap9 gene (from AAV9 serotype) was generated as previously described for the Cap2 gene [START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF]. Briefly, the wild-type Cap9 gene (provided by the Penn Vector Core, University of Pennsylvania, Pennsylvania, USA) was amplified by PCR as described [START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF]. A schematic representation of the Cap9 nucleotide modifications is shown in Supplementary Figure 33. The resulting Rep2opt and Cap9opt genes were then cloned in pFastBac Dual plasmid following instructions provided with the Bac-to-Bac baculovirus expression system kit (Thermo Fisher Scientific, Illkirch, France). Briefly, the Rep78/52opt and Cap9opt genes were cloned under the control of the pPolh (polyhedrin) and the p10 baculoviral promoters respectively to produce the pFBD-Rep2/Cap9opt donor plasmid. Constructions harboring GFP or mSEAP cDNAs, inserted between the two AAV2 inverted terminal repeats (ITR), and controlled by the Cytomegalovirus (CMV) promoter, were cloned in pFastBac 1 plasmid to produce the pFBD-GFP or pFBD-mSEAP donor plasmids respectively. The pFBD-Rep2/Cap9opt, pFBD-GFP, and pFBD-mSEAP were each transformed in DH10Bac competent Escherichia coli cells to produce, by transposition mechanism, the pBAC-Rep2/Cap9opt, pBAC-GFP, or pBAC-mSEAP recombinant baculoviral genomes respectively. As recommended by the manufacturer, the pBAC genomes were transfected with Cellfectin II transfection reagent (Thermo Fisher Scientific) into Sf9 insect cell line (Thermo Fisher Scientific) to produce rBAC-Rep2/Cap9opt, rBAC-GFP or rBAC-mSEAP recombinant baculovirus vectors. Sf9 cells were grown either in adherent or in nonadherent conditions in SFX medium (GE Healthcare, Pasching, Austria) without any serum supplementation. Five rBAC clones of each original construction were picked up from viral plaque assay and amplified through two consecutive infectious passages in Sf9 cells. 24 hours after the last infectious process performed in up to 400 ml of Sf9 cell suspension, baculovirus-infected insect cells (BIIC) were generated [START_REF] Cecchini | Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02-to 200-Liter Cultures[END_REF][START_REF] Wasilko | The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus[END_REF] and 1 to 5 ml aliquots at 10 7 BIIC/ml were immediately cryopreserved in 7.5% dimethylsulfoxide solution diluted in previous conditioned SFX medium.

AAV upstream process

A ratio BIIC versus Sf9 cells of 1 to 10 4 (v/v) was used to initiate the production of AAV vectors. This ratio was described by Cecchini et al [START_REF] Cecchini | Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02-to 200-Liter Cultures[END_REF] as the best producer cell ratio for inducing cell-cycle arrest at the optimum cell density. For the upstream process, the insect cells were cultivated in non-adherent conditions in serum-free SFX medium. Research-scale productions (from 20 ml to 2L) were performed using 125 ml to 3l Erlenmeyer flasks with vented cap (Corning, Amsterdam, The Netherlands) placed in agitator incubators warmed at 26 °C and set to 110 rpm rotary shaking. Pilot-scale productions (5L) were performed in "wavetype" Biostat Cultibag RM bioreactors (Sartorius AG, Göttingen, Germany) set to 20 rocks per minute, 26 °C, 7% rocking angle and 450 ml per minute of air flow rate injected into the culture bag. Seventy-two hours post infection, the culture was stopped and prepared for downstream process.

AAV downstream process

The clarification step was initiated by insect cells disruption using 0.5% final concentration of Triton X-100 detergent (in both Erlenmeyer and bioreactor systems).

RNAse A (Sigma-Aldrich, Saint-Quentin Fallavier, France) was added at a final concentration of 5 µg/ml and the culture was maintained at 37 °C for 1 hour under shaking. For research-scale productions (20 ml to 2L), the suspension was first centrifuged at 3,500 × g for 20 minutes before filtration through a 0.45 µm polyethersulfone membrane syringe filter unit of 33 mm diameter (Millipore, France) for productions up to 100 ml, or a 0.8-0.45 µm double layer polyethersulfone membrane filter of 17.3 cm 2 (Sartorius AG) for productions up to 2L. For 5-L pilot-scale productions, a 20 µm polypropylene prefilter membrane of 500 cm 2 (Sartorius AG) was first used to retain large debris before the use of a 0.8-0.45 µm polyethersulfone filter of 300 cm 2 of filtration surface (Sartorius AG) to finalize the clarification step.

For volumes greater than 400 ml of cultures, a step of concentration by TFF was performed through a polyethersulfone membrane hollow fiber unit with 500 kDa molecular weight cut off placed on an automated Kros Flow Research IIi TFF system (Spectrum Laboratories, Breda, The Netherlands). 115 to 490 cm 2 filtration surfaces were used for 400 ml to 2L productions respectively, and a 1,550 cm 2 filtration surface was used for the 5L productions. A 20× concentration factor was reached in both cases, leading to a final volume of 60 and 240 ml respectively (refer to Table 5).

The purification step was performed by an isopycnic ultracentrifugation which was slightly modified from the previously described protocol [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. What we defined as "classical or conventional process" in the text was performed by filling 16 ml of viral suspension through a glass Pasteur pipette in the bottom of 25 × 89 mm Quick-Seal Polyallomer dome-top tube (Beckman Coulter, Villepinte, France). This suspension was raised up by successive addition of 9 ml of 15% iodixanol (from 60% stock solution; Sigma-Aldrich) at 1M NaCl, 6 ml of 25% iodixanol, 5 ml of 40% iodixanol, and 3 ml of 60% iodixanol. Iodixanol solutions were all diluted in 1× PBS-MK buffer (1M NaCl, 1 mmol/l MgCl2, 2.5 mmol/l KCl, 1× PBS).

Tubes were heat-sealed with the Tube topper system (Beckman Coulter), placed in Ti70 rotor, and ultracentrifuged at 70,000 rpm for 80 minutes in an Optima L100XP ultracentrifuge (Beckman Coulter). The Fraction Recovery system (Beckman Coulter) was used to puncture the tube from the bottom side and the six first milliliters, containing viral particles, were collected. Our "new process" proposed an optimization of the previous method by adding up to 30 ml of the viral suspension in the centrifuge tubes before addition of 3 ml of 25% iodixanol, 4 ml of 40% iodixanol,

In vitro assays and FACS analyses

In vitro assays were performed in 24-well tissue culture plates having 2 cm 2 of growth area per well. 2 × 10 5 HEK293T cells per well were seeded in total volume of 300 µl of 1× Dulbecco's modified Eagle medium supplemented with Glutamax, 4.5 g/l of D-Glucose (Thermo Fisher Scientific), 10% of fetal bovine serum (Biowest, France), and 1% of Penicillin-Streptomycin solution (Life Technologies). Twenty-four hours later, an average of the number of cells per well was determined by trypsinization and automatic cell counting. Vector transduction was performed with rAAV2/9-GFP at logarithmic multiplicity of infection ranging from 10 to 10 4 . Forty-eight hours later, cells were observed by inverted fluorescent microscopy (Leica Microsystems, Nanterre, France) to verify the efficiency of AAV transduction through GFP expression. For quantitative measures, the cell monolayers were first trypsinized and centrifuged at 600 × g for 5 minutes. The pellet was resuspended in 4% paraformaldehyde solution and fixation was done for 10 minutes at room temperature. After centrifugation (600 × g/5 minutes), the fixed cells were homogenized in 1× PBS supplemented with 1% fetal bovine serum. Determination of the percentage of GFP-positive cells was performed by flow cytometry using the ImageStreamX Mark II Amnis Imaging Flow Cytometer (Merk Millipore, Darmstadt, Germany) through the INSPIRE and IDEAS software for acquisition and analysis operations respectively.

In vivo assays

All studies on mice were conducted in strict accordance with the institutional guidelines for animal research transposed from the European directive 2010/63/EU. Project authorization, including all the procedures, was submitted to the Ministry of Higher Education and Research on 31 March 2014. All injections were performed after inhalational anesthesia using isoflurane, and all efforts were made to minimize animal number, procedure lengths, and suffering. Ten-week-old C57BL/10 mice (3 animals per group) were injected intramuscularly with 50 µl of AAV2/9-mSEAP vectors issued from the new purification process. A total of 2.0 × 10 12 vg was injected in TA muscles.

Fifty microliters of blood were withdrawn every week for 1 month, and serum was isolated by blood centrifugation at 10,000 × g for 5 minutes at 4°C. Mice were sacrificed 1 month postinjection by cervical dislocation and TA muscles were harvested. TA muscles were immediately frozen in liquid nitrogen-cooled isopentan solution and cryopreserved at -80 °C.

Ten-week-old C57BL/10 mice (3 animals per group) were also injected intravenously through retro-orbital delivery of 1.0 × 10 13 vg per gram of body mass. To enhance biodistribution, the vectors were diluted in PBS for a final injection volume of 100 µl. A unique blood collection (50 µl) was performed at day 7 and mice were sacrificed 1 month postinjection. TA, triceps, heart, diaphragm, kidney, and liver were immediately frozen as previously described and cryopreserved at -80 °C. Serial 10µm cryosections were made and examined for alkaline phosphatase enzymatic activity. Histological slides were first fixed with 0.5% glutaraldehyde solution for 15 minutes at room temperature followed by 3 cycles of 15 minutes washing with PBS.

To inactivate endogenous alkaline phosphatase activity, slides were incubated at 65°C for 30 minutes in PBS, followed by two PBS washings. 5-Bromo-4-Chloro-3 Indolyphosphate/Nitro Blue Tetrazolium solution (Sigma-Aldrich) was added to cryosections and incubated at 37 °C for 30 minutes to several hours until occurrence of significant visual colored staining, compared to slides of noninjected muscles.

Quantification of mSEAP from mouse serum was performed with the Phospha-Light SEAP Reporter Gene Assay System (Thermo Fisher Scientific) according to manufacturer instructions. Briefly, serum was first 50-fold diluted in a dilution buffer provided with the Phosphalight kit before endogenous alkaline phosphatase inactivation at 65°C for 30 minutes. After sixfold serial dilutions in assay and reaction buffers containing chemiluminescent substrates and enhancers, microplates were placed in a luminometer for luminescence detection. Raw data were analyzed through simple spreadsheet and quantified using using a standard curve previously prepared by serially diluting stock enzyme.

Statistical analyses

Graphical representations are presented as mean ± standard error of means, and a two-tailed Student's t-test is used to determine significant differences in percentage of GFP-positive cells between iodixanol fractions 0-4.5 and 4.5-6 ml (Figure 28g). P < 0.05 represented by * in Figure 28g, is considered as significant. 5. Detailed comparison between classical and new AAV downstream processes. As described in figure 27, the difference was principally due to the optimization of the purification stage in which modification of isopycnic iodixanol gradient construction lead to a significant decrease in process duration and an increase in processed volumes. Importantly, one working day (6h00) was sufficient to treat a five-liter bioreactor producing more than 10 14 viral genomes (vg). Further details on the downstream process are described in material and methods. ND: not determined.

Supplementary results

Table 6. Titers and yields analyzed from rAAV productions performed at 100 ml, 400 ml et 5 liters. The productions correspond to those illustrated in Figure 29a. The yields in vg/ml were determined from the final Sf9 cell concentrations. The yields in iu/ml were determined from the results of the FACS analyses (Figures 28c and31), only performed with the 100 ml productions (ND: not determined).

1 100 ml 1,77E+13 0,225 3,98E+12 3,10E+06 1,30E+04 4,20E+11 2 2,52E+13 0,225 5,66E+12 3,50E+06 1,62E+04 3,40E+11 3 2,58E+13 0,225 5,80E+12 4,00E+06 1,45E+04 6,

III. Genome editing for Duchenne Muscular Dystrophy

We aimed to use the viral vector technology that we dispose in our laboratory in the context of genome editing for muscular diseases, particularly for DMD. As mentioned before, several studies successfully recovered part of the expression of truncated dystrophin in DMD muscles through many methods (allele complementation, exon-skipping, genome editing mediated by non-homologous end-joining-NHEJ). In these approaches, the expressed truncated dystrophin may present a long-term instability as described in some BMD phenotypes. In addition, most of these corrections (except for gene editing by NHEJ) are performed at the RNA level, and if vectors are lost, the disease phenotype may re-appear. Here, in order to circumvent these problems, we attempt to correct the mutation by homology directed repair (HDR) at the genomic level to stably re-express the fulllength dystrophin using the CRISPR-Cas9 system.

CRISPR-Cas9 tools

In order to induce homologous recombination, we designed two plasmids: the first plasmid, named crmdx, carries the sgRNA and the Cas9 derived from One of the important parameter concerns the design of the initial AAV constructs. The choice of the transgene promoter is a central element both in terms of cell or tissue specificity, as well as level of expression or timeline. The use of promoters whose activation is cell-specific makes it possible to direct the expression of transgenes in targeted tissues. Similarly, the level of expression of a transgene can be modulated by the choice of the promoter as a function of whether it is a constitutive promoter recognized for its high level of activation or its natural promoter. In this case, the genetic construct avoids overexpression of the transgene which may have undesirable effects within the targeted tissue. For instance, The U7snRNA transgene, used in exon skipping approaches, is directed by a PolIII promoter. This limits the use of stronger PolII promoters, but allows expression of physiological levels of the modified U7snRNA in quantitative and qualitative manner. Finally, it is possible to control the moment of expression of the transgene of interest according to the stage of development of the targeted organism.

The quality and the integrity of the plasmid constructions used in tritransfection protocols is an important parameter for an optimal production. The maxi-preparations of the plasmids have to be correctly and rigorously controlled before their use in transfection protocol. Their final formulation and restriction profile have to be previously established. Sometimes, we strongly recommend confirming their integrity by sequencing analyzes before to start any transfection. These last remarks, which at first glance appear to be of a technical nature, are not always rigorously respected and can often lead to poor rAAV yields.

The design of molecular constructions is much more problematic in the context of the baculovirus / insect cells production system. It is indeed known that the level of expression of genes and their corresponding protein is linked to the cellular environment in which this expression is performed. In this context, the most important characteristic is linked to the choice of codons more favored by a particular organism. The redundancy of the genetic code allows the use of several base triplets for the same amino acid. The evolution of the living organisms has favored the use of certain codons to the detriment of others according to the place of the species in the phylogenetic tree. This parameter must now be taken into account in order to increase the levels of expression of certain proteins in cellular environments where they are not naturally produced. This is the case for a large part of the recombinant proteins expressed in insect cells. In our case, the genes encoded for the rAAV components (the rep and cap genes) have been codonoptimized to be efficiently expressed in insect cells. In addition, unlike the wild type AAV genome, the rep and cap genes had to be "separated" to be expressed via two strong baculovirus promoters, the polyhedrin (Polh) and the P10 promoters. This molecular "separation" of the two AAV genes was necessary because their natural promoters (P5 and P19 for rep, and P40 for cap) are not (or very poorly) recognized or activated by the transcriptional machinery of the insect cells. On the contrary, it is evident that the sequence of the transgenes of interest, their promoter and possibly their poyladenylation sequence (together flanked by the two ITRs) need not undergo codon optimization if they are to be transcribed and translated into higher mammalian cells (since they correspond to the types of organisms in which they naturally express themselves). Technically, the codon optimization requires approaches of artificial mutagenesis or of gene syntheses.

I.2 Optimization of the rAAV production process

As mentioned in the previous chapter, quantity, purity and quality of rAAV are of extreme importance when these are meant to be employed in gene therapy.

Here, we were able to show new biotechnological improvements for rAAV production both at research scale [START_REF] Dias Florencio | Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors[END_REF] and pilot scale for subsequent studies in large animals and in the perspective of clinical trials [START_REF] Buclez | Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system[END_REF]. In these works, we were able to not only improve the yields and quality of vectors, but also to reduce the cost and the duration of the rAAV production.

This part will focus on the optimization that can be done at the upstream process level. At the research scale, tri-transfection of HEK293 cells is known to be the most widely used production method. It should be recalled that the AAV vectors require helper genes from other large viruses (Adenovirus, Herpesvirus, Papillomavirus,…) to activate their replication. In this context, HEK293 cells have been chosen as producing cells because of the genomic integration of one of the most important helper gene for the replication of AAVs: the adenoviral E1 gene.

Historically, the use of a wild-type adenovirus (as a helper virus) during the production phase of AAV vectors posed both safety problems for manipulators, but also required vigilance and drastic quality control in order to ensure its complete elimination from the final product [START_REF] Xiao | Production of high-titer recombinant adenoassociated virus vectors in the absence of helper adenovirus[END_REF]. Since the identification of the minimal number of adenoviral helper genes required for AAV replication in HEK293 cells, the production of a helper plasmid containing all of these helper genes has been successfully done, and consequently the problem of the wild type adenovirus has been resolved.

In the context of the tri-transfection process, the nature of the transfectant agent used to penetrate the plasmids into the HEK293 cells may also influence its effectiveness. In our laboratory, PEI has been shown to be an effective transfection agent, but other products may possess more efficient transfection properties, notably because of the low cytotoxicity they may present. This may also represent another parameter to be investigate in order to improve and / or simplify the production yields.

During the production phase, AAV vectors synthesized by HEK293 cells can either accumulate in the intracellular compartment or be excreted into the extracellular supernatant. This parameter can greatly influence the production yields depending of the fraction that will be treated during the subsequent purification steps. This will be described in detail in the next section.

The amounts of AAV vectors produced by the tri-transfection system are limited by the adherent characteristic of the producing cells, and therefore require large surfaces of culture to achieve the particle concentrations required for preclinical or clinical assays. To circumvent this crucial inconvenient, other production systems have been implemented: one of the most efficient system derived from the baculovirus expression vector system (BEVS).

BEVS is a system used for many decades to produce recombinant proteins on an industrial scale. In 2002, Robert Kotin's team decided to test this method to produce rAAV vectors, which confirmed the possibility of using baculovirus to significantly increase the levels of production of these vectors and thus be able to meet the needs of clinical trials [START_REF] Urabe | Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors[END_REF]. The original production system used three baculoviruses recombinant for AAV genes: rep, cap and the transgene of interest (flanked by the ITRs). Indeed, this system does not require the addition in trans of helper genes as for the tri-transfection system. The helper functions necessary for the mobilization of the AAV genome are probably provided directly by the environment of the insect cell with a hypothetical contribution of both cellular and baculoviral genes. The identification of the helper functions in the insect cell environment deserves to be investigated in depth and could contribute to significant developments in the BEVS system.

The 3 infection method has evolved these last years, notably in reducing the number of recombinant baculoviruses used for production of rAAV. The most widely used system currently relies on infection of insect cells with only two recombinant baculoviruses, the first carrying both the rep and cap genes and the second the transgene of interest (flanked by the ITRs). Single infections were performed by the use of stable insect cell lines which previously integrate the transgene construction into their genome. Nevertheless, this kind of approach has to be ameliorate because of the difficulty to obtain stable insect cell lines in vitro.

Our laboratory is actually working on the use of one single recombinant baculovirus harboring all of the elements necessary for the production of rAAV.

Moreover, in the context of process optimization, it has been found important to solve the problem of the cryo-conservation of baculoviruses for successive productions. Indeed, recombinant baculoviruses cryopreserved poorly (decrease in viral titres after thawing). To solve this problem, Wasilko et al [START_REF] Wasilko | The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus[END_REF] proposed an alternative that consisted of directly cryo-preserving the insect cells previously infected with baculoviruses: these cells are named BIIC (Baculovirus-Infected Insect Cells). In this case, cryo-preservation should occur at the beginning of the viral cycle and before the initiation of the lytic activity. This method makes it possible to better protect the baculoviruses from potential degradation and thus to directly use the BIICs in co-culture with virgin insect cells (i.e. Sf9) to initiate a new rAAV production. Concretely, the re-culture of the BIICs re-initiates the baculoviral cycle in the intracellular compartment. Recombinant baculoviruses from the BIICs finally exit into the supernatant and initiate new cycles of infection in a chain reaction that typically ends 72 hours to 96 hours later when all of the Sf9 cells of the initial culture have been infected. The great advantage of this method is the BIIC / Sf9 ratio used for the productions. In our hands, this ratio was optimized to be equivalent to one volume of BIIC for 10 4 volumes of Sf9 cells, meaning for example that for an initial 10 liters volume of culture one single milliliter of BIIC is sufficient to perform an optimal rAAV production. Knowing that we produce on average almost 100 ml of specific BIIC by amplification, we theoretically have raw materials to generate more than 1000 liters of productions. These volumes start to correspond to industrial volumes.

I.3 Optimization of the rAAV purification process

The downstream process is certainly the part of the process in which the yield losses are the most important, and which consequently requires the most optimization efforts. It is well known in the world of biotechnology that every purification step undoubtedly leads to material losses. One of the development activities consists of limiting these losses of material by a judicious choice of the successive methods employed as well as of the nature of the material used.

Moreover, from an industrial perspective, it is also important to worry about the financial impact and the time devoted to the downstream process. The transfer of technology from the academic to the industrial world will be facilitated if all the characteristics described above are optimized to match with this objective. In the perspective of pre-clinical and future clinical gene therapy trials we have endeavored to develop methods of rAAV vector production compatible with the quantitative and qualitative requirements of these assays.

Through two successive publications, we focused our efforts in the goal of efficient, fast and inexpensive purification processes [START_REF] Buclez | Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system[END_REF][START_REF] Dias Florencio | Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors[END_REF]. These developments were provided at all phases of the downstream process: clarification, concentration, purification and polishing.

Clarification step

The clarification corresponds to the first step of the downstream process and allows the elimination of the large "contaminants" often represented by cellular debris and other macroscopic aggregates issued from the crude lysate.

As part of our optimization of production processes at the research scale (tritransfection protocol), we were led to compare existing protocols with the one we wanted to implement. As briefly mentioned above, the rAAV vectors produced in the HEK293 cells are located at the end of the process either in the intracellular compartment or excreted in the supernatant. To avoid manipulating too large volumes of production, one of the most commonly used approaches is to focus only on the cellular part of the culture (cell pellet) and to ignore the "supernatant" part.

In this context the purification protocol goes through a series of methods including a freeze / thaw cycle of the cell pellet to release the AAV particles which is very time consuming. Moreover, this step is hypothetically not without consequence on the integrity of the viral particles as will be discussed in a later part.

Other protocols describe the possibility of harvesting rAAVs only from the culture supernatant. The disadvantage of this approach lies in the treatment of large volumes of culture. This issue will be discussed in the next section. However, it emerged from these studies that cell supernatants also contained a large portion of AAV vectors. It then seemed obvious to us to find a method that can both treat the rAAV particles contained in the cells and in the supernatant.

For this we used an anionic detergent, Triton X-100, known for its ability to solubilize cell membranes without altering the protein content of the crude lysate.

This approach allows us to recover in a single step both the rAAV content from the cells and the supernatant. This method is a significant time saving compared to the The approach described for tri-transfection clarification is also used successfully to treat the productions made by the BEVS system.

Concentration step

As briefly mentioned in the previous section, the generation of large volumes of production raises the question of their treatment and especially of their concentration. This is true both for the BEVS system and for the treatment of the whole cell culture (cell pellet and supernatant) resulting from the tri-transfection protocol. A well-known method in the industrial world is to significantly reduce production volumes by tangential flow filtration (TFF). This approach is now successfully applied in our laboratory for both types of production systems. It allowed us to optimize it for rAAV vectors by playing both on the cutoff thresholds and on the nature of the membranes used. In addition, the introduction of fully automated TFF instruments, allowing fine adjustment of the pressure and liquid flow parameters, provides significant safety, comfort and time savings to the process. More concretely, the use of TFF allows us to reduce the volumes of clarifiates by 20 to 50 times depending on the initial volumes to be treated. These levels of concentration allowed us to implement an efficient and inexpensive purification process as described below.

Purification step

The key step in the downstream process is the purification. For rAAV vectors, there are different approaches that should be listed to better understand our choices. The concept of purification in biotechnology is often associated with chromatography techniques. Immunoaffinity, ion-exchange and hydrophobic interaction chromatography is very often the solution favored by industrial approaches. Briefly, affinity chromatography techniques are based on a reversible interaction between the viral capsid and a specific ligand coupled to a chromatographic matrix [START_REF] Burova | Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications[END_REF] . This specific interaction will allow the separation of viral particles from its contaminants (protein and DNA), that are not able to bind the matrix. In particular, for rAAV purification the most commonly used ligands are llama antibodies [START_REF] Wang | Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin[END_REF]. This purification method has shown very interesting interactions and reversibility with rAAV capsids, however, one limitation of this method is that the ligand is usually serotype specific. In addition, elution solutions, generally low pH or high ionic strength solutions are used to recover the AAV particles and may damage the capsid, which will lead to lower quality of the final product. Purification by ionexchange chromatography is based on the charge of proteins presents on the viral capsid, that depends on the pH of the exposed amino-acid groups. Both cationic and anionic exchange chromatography techniques are used and the AAV surface charge may vary among serotypes, which leads to variable results concerning the production efficacy [START_REF] Potter | A simplified purification protocol for recombinant adeno-associated virus vectors[END_REF]. Hydrophobic interaction chromatography is based on binding of the viral capsid proteins to the column matrix through hydrophobic interaction in an aqueous solvent. A salt at high concentration is used as a solvent to promote clustering of hydrophobic protein surfaces, thereby minimizing their exposure to solvent. However, high salt concentration may destabilize viral particles, resulting in viral degradation.

In addition, chromatography techniques, and more particularly the affinity chromatography, are very costly approaches because of the price of the stationary phases media. Moreover, these approaches do not discriminate between full and empty particles, both generated during the upstream process. To circumvent these problems, we chose to evaluate a physical purification approach based on the sedimentation rate of rAAV particles. We opted for an ultracentrifugation on a discontinuous gradient of iodixanol in order to allow the rAAVs of interest to stabilize at their equilibrium density level. The absence of a genome in the empty particles gives them a lower equilibrium density than the full particles. The optimizations we have made compared to the protocols described in the literature have enabled us not only to significantly reduce the time of the purification process but also to be able to process much larger volumes of production. Moreover, unlike chromatographic methods, the iodixanol discontinuous gradient ultracentrifugation approach allows an efficient separation of the full and empty particles. Five liters of crude lysate production were processed by a single round of ultracentrifugation, thus allowing the implementation of this technique for much larger volumes, certainly compatible with industrial requirements.

Polishing step

The polishing step corresponds to a step of removing some residual contaminants and reformulating the vectors in their final buffer. It is often associated with separation techniques based on the physical particle size. The best known polishing approach is the gel filtration (also known as exclusion chromatography) and uses a stationary phase consisting of porous beads capable of slowing the migration of some constituents to the detriment of others of larger size; with a judicious choice of the pore size, it is possible to achieve a maximum purification of the vectors by minimizing the loss of yields. Gel filtration columns designed for the separation of particles of sizes equivalent to the rAAV vectors are now commercially available and are being evaluated in the laboratory to further optimize the final quality of the productions.

Reformulation of the products resulting from the purification steps is often carried out by TFF or dialysis systems. The former is preferred over the latter because of their speed of execution. However, TFF systems pose problems of yield losses which must be taken into account at the end of process.

The quality controls carried out during our process development allowed us to appreciate both the final qualities of our rAAVs and also the large quantity of vectors recovered. These results logically led us to evaluate them for their functionality under in vitro and especially in vivo conditions, as described below.

I.4. Comparison between the new method described (Buclez et al., 2016) and the classical method (Cecchini et al., 2011)

The classical BIIC-mediated rAAV production was described in 2011 by Cecchini and colleagues. Here we compare this classical method with our new purification method described in [START_REF] Buclez | Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system[END_REF]. The upstream method used in both production techniques remains the same, the downstream method is however slightly different. Following 72h of upstream process, the classical method downstream processing starts with a physical cell lysis with a mechanical cell disrupter and a subsequent nuclease treatment which includes both DNase and RNase. In our new protocol, we decided to perform in a single step a chemical cell lysis with Triton X-100 and a RNase treatment, eliminating the DNase cost prohibitive step. The Triton X-100 treatment, previously used in our rAAV purification following tri-transfection in Dias-Florencio et al., 2016, is capable to solubilize cell membranes without altering the protein content of the lysate, which offer protection to the rAAV capsids. Because of the higher quantity of contaminants and debris following this chemical treatment, a pre-filtration through a 20µm membrane is required. In contrast, the classical method performs the prefiltration through a 1,2µm membrane before the common step of filtration through 0,8µm and 0,2µm membranes. The classical method continues with an affinity chromatography step to purify the lysate, and a subsequent concentration step with tangential flow filtration (TFF). The new method, on the other hand, performs first a concentration method, which allows us to purify the lysate using discontinuous iodixanol gradient coupled with ultracentrifugation. As mentioned before, the suppression of this chromatography step allows us to reduce the production cost.

In addition, the ultracentrifugation offers the possibility to separate empty and full particles, as well as the elimination of DNA contaminants that may still be present in the lysate. The adjustment of the volumes for the discontinuous iodixanol gradient phases to 2 ml, 4 ml and 3 ml instead of 5, 5, and 6 ml for the 60, 40, and 25% phases, respectively allow us a two-fold increase in the volume treated in a single run 20 minutes run compared to the original publication [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. With these adjustments, a Ti70 rotor type we can now treat up to 240 ml of viral suspension in just 20 minutes. If this volume of viral suspension derived from a usual 30 to 40 times concentrated solution following TFF, it then turns out that a single 20 minutes run of ultracentrifugation is able to process a starting raw production of approximately 10 l. In an industrial context, the implementation of series of ultracentrifuge systems to easily multiply the level of purification capacity offered by our new iodixanol gradient approach.

Both downstream processes finishes with diafiltration accompanied by sterile filtration. Of note, the diafiltration step, using in this new method centrifugal filtration units as reformulation systems, can easily be replaced by TFF systems to be more compatible with industrial requirements.

In conclusion, the baculovirus/insect cell system confers ultimately the possibility of scaling up the levels of production much more than can be provided by processes using HEK293T adherent cells. The methods described above represent reproducible methods that can be easily incorporated in research laboratories and industrial scale for the flexibility in volumes of production.

I.5. Improvement of the rAAV transduction efficiency

The transduction efficiency of the rAAV vectors remains the main evaluation step after the optimizations of the production and purification processes. The results issue from our research-scale developments (tri-transfection protocol) have led us to note, with some surprises, the efficacy of the vectors derived from our new method compared to those issue from other methods. The first advantage is quantitative: collection of the rAAV vectors from both the intracellular compartment and the supernatant allows us to significantly increase the number of viral particles relative to the only cellular or supernatant collection protocols. We thus confirm that it is necessary to implement a method allowing a global collection of rAAV vectors: this approach, carried out in the early stages of the downstream process (clarification step), allows a significant improvement in rAAV yields compared with other methods conventionally used. Beyond these quantitative considerations, the results obtained with the in vitro and in vivo transduction experiments allowed us to observe a better gene transfer efficiency with the rAAVs resulting from our new process.

To explain the better functionality of vectors issued from the new method, one hypothesis is that our new process offers less damaging conditions for the vectors.

Indeed, the drastic physical/chemical treatment used to induce cell lyses to recover vectors from cell pellet may impact the quality of rAAV capsids. Moreover, vectors that are released during the 72h period, can be confronted to a progressive acidification of the culture medium resulting from cell metabolism and action of proteases, which could induce structural alterations. The fact that the supernatant recovered vectors method take in charge only this fraction of viruses may negatively impact the quality of the final product. The results provided by this work suggests that the detergent (Triton X-100) may provide a protective treatment to the structure of vectors, and allows the suppression of damaging techniques to induce cell lysis, that could in fact alter the quality of capsids.

The same qualitative effects were obtained with the rAAV vectors from the BEVS system. The quantitative difference with the tri-transfection approach allowed us to observe the ability of the rAAV vectors (serotype 9) to bio-distribute very efficiently throughout different tissues after their systemic injection. These encouraging results reinforce the idea that efficient development of production and purification processes, capable of ensuring both high concentrations and high quality of rAAV vectors, is a promising avenue for implementing the future preclinical or clinical protocols of gene therapy. To correctly identify these targeted cells, one must develop a co-staining with a mSEAP-specific antibody and neuronal cells (neurons, interneurons, astrocytes, microglia) specific antibodies, which we were not able to put in place so far. Some hypothesis can be formulated. Even though staining is mostly spread without any organization, one area in particular catches our attention. Analyzing the mSEAP staining in the hippocampus (figure 34) we can identify an orderly staining forming a line between the dentate gyrus and Cornu Ammonis 1 (CA1). The hippocampus Moreover, other hypothesis can still be envisaged. Since this staining is punctual and non-specific to any brain region, these purple dots transduced by rAAV2/9 could be endothelial cells from blood vessels. This could open the doors to a whole other group of diseases affecting the brain vascular system. For example, cerebroretinal vasculopathy [START_REF] Grand | Cerebroretinal vasculopathy. A new hereditary syndrome[END_REF], Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [START_REF] Joutel | Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia[END_REF] or Cerebral small vessel disease (SVD) [START_REF] Choi | Genetics of Cerebral Small Vessel Disease[END_REF] are diseases with no effective treatment today that could beneficiate from a specific targeting of AAV9 with no invasive approaches.

I.7. Clinical perspectives challenges of rAAV vectors

The history of gene therapy is tainted with some cases of negligence regarding guidelines which elicited several ethical debates. Premature testing of viral vectormediated therapy resulted in the stigmatization of this technology. This is, however, one important step toward the treatment of several patients with genetic diseases that still today have no treatment or cure. Viruses offer clever mechanisms to promote cellular internalization of a therapeutical transgene, which is still very limited for non-viral mediated-therapies. Nevertheless, viral vectors are still confronted with many challenges before becoming a democratized and widely used tool for gene therapy. One of the major problems today is still the safety of viral vectors injections regarding patient's immune system. Patients immunity play a major role in the acceptance of the vectors by the body, and the premature activation of the immune system with the production of neutralizing antibodies against viral capsids impacts directly the efficacy of the treatment. The immune response against viral vectors can be cytotoxic or humoral, by the production of antibodies [START_REF] Mechnikov | Immunity In Infective Diseases[END_REF]. In the case of rAAVs, the immune response is mainly humoral and the seroprevalence in the human population can achieve rates up to 60%, which can lead to the rejection of injected therapeutical vectors. This is a limiting point to the use of this technology. A couple of strategies can be engaged to evade this established acquired immunity: one of them is capsid engineering.

The rational-design of AAV capsids may alter AAV-host interaction by the modification of capsid epitopes previously known to be recognized by the immune system. This strategy can be used not only to evade the immune system, but also to favor specific cell targeting by enforcing virus-receptor and/or co-receptor interaction. Random mutagenesis is being developed in order to generate a large AAV serotype library. Three strategies can be used to induce random mutagenesis:

(1) Error prone, a PCR based mutagenesis in which point mutations are randomly introduced in the capsid, leading to several amino acid changes; (2) Capsid shuffling, in which the capsid of several serotypes are assorted to generate new serotypes; and (3) Display, based on the introduction of oligonucleotides on the capsid surface [START_REF] Büning | Engineering the AAV capsid to optimize vector-host-interactions[END_REF]. Following generation, new serotypes of AAV are screened for virus-host interaction and cell targeting. Also, recently studies were able to reconstruct the AAV phylogenetic tree and characterize ancestral AAV serotypes [START_REF] Santiago-Ortiz | AAV ancestral reconstruction library enables selection of broadly infectious viral variants[END_REF]. Today, the human population presents very low or absent seroprevalence for these serotypes and the rational engineering of AAVs with these characteristics can be a useful strategy. The other strategy to evade host immune response is the use of decoy capsids. These are empty AAV capsids that will capture neutralizing antibodies and allow the therapeutical vectors to entry the cells. Moreover, it is a major concern that the production of rAAV generates pure and high quality vectors, avoiding the generation of empty particles and partially packaged vectors that may elicit a cytotoxic reaction. To heighten the safety of these decoy capsids, a group developed engineered decoys that are not able to entry cells, therefore eliminating the possibility of a cytotoxic response [START_REF] Mingozzi | Overcoming preexisting humoral immunity to AAV using capsid decoys[END_REF]. Because of AAV large biodistribution, it is important to invest in these innovative strategies since they represent a powerful tool for gene transfer. The substantial biodistribution is an important characteristic of these vectors because the use of viral vectors in general are still limited to one shot, and the maximum number of cells must be corrected upon the first treatment.

Another characteristic of these AAV virus that may limit the use in certain therapies is that they are one of the smallest virus, which implicate also in a very limited packaging size. With a 4,7kb genome, approximately 3,6kb can be replaced by a transgene. For DMD for instance, this prohibit the delivery of the full-length dystrophin gene, more than 11kb in size. Several studies invested in the delivery of truncated forms of this protein, capable of being encapsidaded by AAVs. Moreover, as already described, it is known that the shorter dystrophin is not as stable as the full-length form of the protein, which may lead fibers to burst if a homogenous reexpression of dystrophin is not achieved. An alternative to the allele complementation by the delivery of the full-length dystrophin is gene editing to repair the mutation directly in the genome. This strategy offers several advantages over allele complementation and even exon-skipping. The permanent repair at genomic level implies life-long efficacy of the treatment. The recently developed gene editing CRISPR-Cas9 system gives high hopes for the treatment of DMD.

Thanks to last developments, the CRISPR-Cas9 system can now be vectorized by rAAVs. Indeed, this system was formerly composed by the SpCas9 from can be effectively exploited in both cycling and post-mitotic cells, such as muscle fibers [START_REF] Saha | Examining DNA Double-Strand Break Repair in a Cell Cycle-Dependent Manner[END_REF]. However, this strategy may induce nonspecific insertional mutagenesis and the generation of internally truncated dystrophin. On the other hand, HDR can only target proliferating tissues during phases S, when cell undergoes DNA replication, and G2, when occurs protein synthesis and cell growth previous to cell division [START_REF] Rothkamm | Pathways of DNA doublestrand break repair during the mammalian cell cycle[END_REF]. Little or no repair occurs by HDR in other phases of cell cycle. This happens because the occurrence of HDR is dependent on the recruitment of specific recombination proteins (Rad52, Rad59, Rdh54, Rad51, Rad55 and Rad54), activated only during phases S/G2 [START_REF] Kostyrko | A role for homologous recombination proteins in cell cycle regulation[END_REF][START_REF] Lisby | Choreography of the DNA Damage Response[END_REF]. Indeed, the post-mitotic nature of the adult muscular tissue together with the challenges offered by the HRD-mediated gene repair, have directed most studies for DMD towards the NHEJ strategy, leading to efficient genomic exon-skipping results [START_REF] Bengtsson | Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy[END_REF][START_REF] Iyombe-Engembe | Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method[END_REF][START_REF] Long | Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA[END_REF][START_REF] Long | Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy[END_REF][START_REF] Wang | The AAV-mediated and RNAguided CRISPR/Cas9 system for gene therapy of DMD and BMD[END_REF][START_REF] Xu | CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice[END_REF][START_REF] Zhang | CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice[END_REF].

However, we decided to study further the possibility of inducing HDR in muscular tissue. In order to stimulate HDR correction in muscle fibers, we first injected cardiotoxin (CTX), a snake toxin that will induce muscle regeneration in wild-type C57BL/10 mice: we thus induce a proliferation process in order to ensure the hypothetical activation of the expression of proteins supporting the homologous recombination process. 72h and 96h later we injected rAAV 2/9 carrying CRISPR-Cas9 transgenes. The template used to induce recombination carries a small modification to replace the PAM sequence, which can be used as a tag for the identification of HDR occurrence. With primers that only recognize this new tag, PCR results showed two bands, in the DNA extracted from CRISPR-Cas9 corrected muscles. The same two bands were previously observed in in vitro experiments, however at a lower rate. The time-line for the injections of AAV-CRISPR-Cas9 tools was chosen to observe the rate of recombination in different steps of tissue regeneration. However, observing the results from the muscular regeneration during four days post-CTX injection, we think that earlier injections would have resulted in higher rates of recombination since at 96h for instance the tissue was almost entirely regenerated. Sequencing results from the bands extracted from in vitro experiments, performed in dystrophic mdx myoblasts, revealed that the higher band, of approximately 500bp, slightly lower than the expected size displayed the corrected sequence and the mutation was in fact repaired. In addition, we could observe the presence of the modified PAM in the genome extracted from these muscle tissues. The lower band sequencing didn't give any exploitable results.

Differences between the sizes expected and obtained may be explained by a difficulty of DNA amplification due to secondary structures. Another hypothesis is a recombination issue that led to the exclusion of a portion of intronic sequences.

These results will be further analyzed with the sequencing of the whole sequence.

Taken together, these results shows that HDR can indeed occur upon induction of a regenerative state in muscles, however, can it occur in the DMD scenario? One perspective to induce homologous recombination in DMD patients is finding the right therapeutical window. Indeed, children musculature is still under development, and especially dystrophic muscle at early phases of DMD pathology goes through several degeneration-regeneration cycles. The pool of satellite cells is recruited to regenerate damaged muscles but this pool is eventually depleted, which leads muscle to undergoes fibrosis and replacement by fat cells [START_REF] Dayanidhi | Skeletal muscle satellite cells: Mediators of muscle growth during development and implications for developmental disorders: Satellite Cells during Growth[END_REF]. When diagnosed early, the treatment of DMD patients by HDR gene editing could be envisaged before the depletion of this satellite cell pool and before severe muscle atrophy, since the dystrophic muscle will be in an almost permanent regenerative state. Further study with neonatal animals should be done to provide evidence of the feasibility of this treatment.

Moreover, recent studies showed that Cas9 expression can elicit host-immune response, with enlargement of lymph nodes, and increased cell counts in mice. This study compared immune response against AAV9 and the Cas9 enzyme and results

showed that while AAV9 elicit humoral response, the injection of Cas9 induced a cytotoxic response with activation of CD4 + and CD8 + cells [START_REF] Chew | A multifunctional AAV-CRISPR-Cas9 and its host response[END_REF].

CRISPR-Cas9 and gene editing technology in general is not free from ethical safety concerns and debates. Indeed, this technology is capable to induce permanent changes in the human genome and the speed with which this technology is going from the labs to the clinic is a bit worrying. The discovery of this technology happened in 1987 [START_REF] Ishino | Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[END_REF], but only in 2012 a study showed that modifications in the system could be performed to target and cleave any DNA sequence [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF]. Since then, substantial improvement has been made in the development of the strategy and the first human trials are already happening. In 2016 the first clinical trial using CRISPR-Cas9 in one patient with aggressive lung cancer received ethical approval by the hospital review board of the West China Hospital [START_REF] Cyranoski | CRISPR gene-editing tested in a person for the first time[END_REF]. This dose-escalating phase I clinical trial aims to induce knock-out of the PD-1 gene in patient's T cells ex-vivo and inject them back to the patient. So far the trial has been considered safe and more patients are being enrolled. The main concern with this technology is the specificity of the modifications that may be introduced by the injection of endonucleases such as Cas9 and the possible off-target effects of this approach that may disrupt other normally functioning genes or even activate oncogenes [START_REF] Cho | Analysis of offtarget effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[END_REF]. Several developments have been made in order to improve specificity of this system with technologies such as CIRCLE-seq [START_REF] Tsai | CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease offtargets[END_REF], that can provide highly accurate detection of targeting sites of the CRISPR-Cas9 system. Furthermore, another major concern is raised: does only somatic cells are edited? Ethical debates are heated when the question is edition of the germline and human embryos [START_REF] Lanphier | Don't edit the human germ line[END_REF]. It is important to keep in mind that the edition of germ cells at the genomic level implies the vertical transmission of the modification to the progeny. The unpredictability of results from editing embryos and the impact this will bring upon next generations are among the important questions raised. Because of this, today, around 40 countries discourage or prohibited the use of gene editing tools in human embryos [START_REF] Lanphier | Don't edit the human germ line[END_REF]. For many, this is the point where we can say science has gone too far. CRISPR-Cas9 developments came a long way in these last 5 years, however, important questions must still be answered before considering this strategy as a safe tool for therapy. • Incubate 20min at room temperature;

• Add the complex to a bottle containing the transfection medium: DMEM 4,5 g/l glucose, + GlutaMAX -pyruvate, 2% SVF, 1% P/S;

• Transfer the medium to the HP slowly

3.HARVESTING AND CELL LYSIS:

3 days after transfection CP method:

• Transfer 100mL of supernatant to a centrifugation tube, shake vigorously the HF until cells detach and transfer all to the centrifuge tube.

• pBAC genomes were transfected with Cellfectin II transfection reagent (Thermo Fisher Scientific) into Sf9 insect cell line (Thermo Fisher Scientific) to produce rBAC-Rep2/Cap9opt, rBAC-GFP or rBAC-mSEAP recombinant baculovirus vectors.

• Sf9 cells were grown either in nonadherent conditions in SFX medium (GE Healthcare, Pasching, Austria) without any serum supplementation.

• Five rBAC clones of each original construction were picked up from viral plaque assay and amplified through two consecutive infectious passages in Sf9 cells.

• 24 hours after the last infectious process performed in up to 400 ml of Sf9 cell suspension, baculovirus-infected insect cells (BIIC) were generated and 1 to 5 ml aliquots at 10 7 BIIC/ml were immediately cryopreserved in 7.5% dimethylsulfoxide solution diluted in previous conditioned SFX medium.

Upstream processes

• Non-adherent sf9 insect cells were cultivated in serum-free SFX medium in two conditions: research scale (2L) in a 3L erlenmeyer and pilot scale (5L) in the "wave-type" Biostat Cultibag RM bioreactors (Sartorius AG, Göttingen, Germany) both at 26°C under shaking.

• Sf9 cells were infected with BIIC at a ratio 1 to 10 4 (v/v BIIC vs Sf9) and culture was ended 72h post-infection.

* Research-scale productions (from 20 ml to 2 l) were performed using 125 ml to 3l

Erlenmeyer flasks with vented cap (Corning, Amsterdam, The Netherlands) placed in agitator incubators warmed at 26 °C and set to 110 rpm rotary shaking.

* Pilot-scale productions (5 l) were performed in "wave-type" Biostat Cultibag RM bioreactors (Sartorius AG, Göttingen, Germany) set to 20 rocks per minute, 26 °C, 7%

rocking angle and 450 ml per minute of air flow rate injected into the culture bag.

Downstream processes

Cell lysis and clarification:

• Clarification of the suspensions with 0.5% final concentration of Triton X-100 and RNAse A (Sigma-Aldrich, Saint-Quentin Fallavier, France) at a final concentration of 5 µg/ml at 37 °C for 1 hour under shaking.

• Filtration:

* For research-scale productions (20 ml to 2 l), the suspension was first centrifuged at 3,500 × g for 20 minutes before filtration through a 0.45 µm polyethersulfone membrane syringe filter unit of 33 mm diameter (Millipore, France) for productions up to 100 ml, or a 0.8-0.45 µm double layer polyethersulfone membrane filter of 17.3 cm 2 (Sartorius AG) for productions up to 2 l.

* For 5-l pilot-scale productions, a 20 µm polypropylene prefilter membrane of 500 cm 2 (Sartorius AG) was first used to retain large debris before the use of a 0.8-0.45 µm polyethersulfone filter of 300 cm 2 of filtration surface (Sartorius AG) to finalize the clarification step.

Tangential Flow Filtration

TFF is performed through a polyethersulfone membrane hollow fiber unit with 500 kDa molecular weight cut off placed on an automated Kros Flow Research IIi TFF system (Spectrum Laboratories, Breda, The Netherlands).

*For volumes from 400mL to 2L: 115 to 490 cm 2 filtration surfaces *For volumes reaching 5L: 1,550 cm 2 filtration surface was used for the 5 l productions.

A 20× concentration factor: final volume of 60 and 240 ml respectively.

Purification (Isopycnic Ultracentrifugation)

• 30 ml of viral suspension through a glass Pasteur pipette in the bottom of 25 × 89 mm Quick-Seal Polyallomer dome-top tube (Beckman Coulter, Villepinte, France)

• Addition of 3 ml of 25% iodixanol (from 60% stock solution; Sigma-Aldrich) at 1M NaCl, 4 ml of 40% iodixanol, and 2 ml of 60% iodixanol. Iodixanol solutions were all diluted in 1× PBS-MK buffer (1M NaCl, 1 mmol/l MgCl2, 2.5 mmol/l KCl, 1× PBS).

• Tubes were heat-sealed with the Tube topper system (Beckman Coulter), placed in Ti70 rotor, and ultracentrifuged at 70,000 rpm for 20 minutes in an Optima L100XP ultracentrifuge (Beckman Coulter).

• The Fraction Recovery system (Beckman Coulter) was used to puncture the tube from the bottom side and the first 4,5mL, containing viral particles, were collected.

• Diafiltration was performed three times through centrifugal concentration and filtration unit Amicon Ultra-15 (Millipore, Guyancourt, France), leading to a 450 times dilution factor. • Taqman Q-PCR is performed by using the iTaq Universal Probes Supermix (BioRad):

-0.1 µmol/l of AAV22mers forward primer (5′-CTCCATCACTAGGGGTTCCTTG-3′) -0.3 µmol/l of AAV12mers reverse primer (5′-GTAGATAAGTAGCATGGC-3′) -0.1 µmol/l of AAV_MGB.P Fam Taqman probe (5′-TAGTTAATGATTAACCC-3′, Life Technologies).

• Fifteen microliters of the previous Taqman mix are added to 10 µl of diluted AAV DNA.

•

In parallel, 10 µl of serially diluted AAV plasmid, containing ITR2 sequences (from 10 8 to 10 2 copies per well) is deposited in duplicates to serve as plasmid range.

• PCR is run in CFX96 Touch Real-Time PCR Detection System (BioRad) under following conditions: hold at 95 °C for 15 minutes, and 40 cycles at 95 °C/15 seconds (denaturation) and 54 °C/1 minute (hybridization).

• Raw data were exported and analyzed by the BioRad CFX manager software.

Titration results are given in viral genomes/ml (vg/ml). -After migration, proteins and nucleic acid content was revealed by using Silver Staining Plus protocol (Bio-Rad Laboratories) following manufacturer instructions.

PT04. AAV purity

-

The gel was deposited on white tray for picture acquisition by Gel Doc EZ system and analyzed by the Image Lab software (Bio-Rad).

PT05. Cell culture of C2C12 cells (immortalized mouse myoblast cell line), and primary C57BL/10 and mdx myoblasts.

1) Muscle tissue harvest and digestion

Note: use mice between 1 to 4 weeks of age (juvenile) or between 5 to 10 weeks of age (adult). Yields of primary myoblasts will be significantly reduced if using older mice. This protocol is based on muscles from one mouse. For multiple mice, muscles from each mouse should be treated in separate dishes.

Material

III. 1 . 2

 12 Genetic and differences between Becker muscular dystrophy and Duchenne muscular dystrophy ........................................................................................................................... III.1.3 Animal models ........................................................................................................................ a. mdx mouse .................................................................................................................................. b. Double knockout dystrophin/utrophin (dKO or mdx/utrn -/-) ........................................... c. mdx52 ........................................................................................................................................... d. Dmd-mutated rats ...................................................................................................................... e. Canine model (GRMD) ............................................................................................................ f. Pig model .....................................................................................................................................

  FIGURE 1. HORIZONTAL GENE TRANSFER IN PROKARYOTES: THE TRANSFORMATION AND CONJUGATION PROCESSES. ................................................................................................................... FIGURE 2. ELECTROPORATION MEDIATED DNA UPTAKE BY RECIPIENT CELLS. ........................................ FIGURE 3. CELL-ENTRY MECHANISM OF DIFFERENT NON-VIRAL VECTORS. ............................................... FIGURE 4. HERPES SIMPLEX VIRUS STRUCTURE. ........................................................................................... FIGURE 5. LENTIVIRUS STRUCTURE.. ............................................................................................................. FIGURE 6. ADENOVIRUS STRUCTURE. ............................................................................................................ FIGURE 7. ADENO-ASSOCIATED VIRUS STRUCTURE ..................................................................................... FIGURE 8. STRUCTURE OF AAV GENOME WITH THE ORGANIZATION OF THE REP AND CAP GENES AND THE RESULTING PROTEINS. ................................................................................................................... FIGURE 9. CLASSICAL RAAV PRODUCTION PROCESS BY THE USE OF TRI-TRANSFECTION METHOD. ...... FIGURE 10. TRANSPOSITION PROCESS TO PRODUCE RBAC AND BIIC ...................................................... FIGURE 11. UPSTREAM PROCESS TO PRODUCE RAAV FROM BIIC. ............................................................ FIGURE 12. CLINICAL SIGNS AND PROGNOSIS OF DMD. ............................................................................ FIGURE 13. THE DYSTROPHIN GENE AND PROMOTERS. ............................................................................... FIGURE 14. DYSTROPHIN AND DYSTROPHIN-ASSOCIATED PROTEIN COMPLEX. ..................................... FIGURE 15. EXON-SKIPPING STRATEGY. ........................................................................................................ FIGURE 16. MINI-AND MICRO-DYSTROPHIN ISOFORMS COMPARED TO UTROPHIN PROTEIN STRUCTURE. ............................................................................................................................................ FIGURE 17. MODIFIED U7SNRNA SHUTTLE SYSTEM. .................................................................................. FIGURE 18. GENE EDITING TOOLS. THE MAIN ENGINEERED ENDONUCLEASES USED IN GENE EDITING . FIGURE 19. THE MECHANISM OF GENOME EDITING USING CRISPR/CAS9. .............................................. FIGURE 20. COMPARISON OF THE THREE DIFFERENT PURIFICATION PROCESSES OF RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV). .................................................................................................. FIGURE 21. COMPARISON OF VECTOR YIELDS AND PURITY BETWEEN CP, SN, AND TC PROCESSES. ...... FIGURE 22. COMPARISON OF IN VITRO TRANSDUCTION EFFICIENCY OF RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) RECOVERED FROM CP, SN, OR TC PROCESSES. ................................. FIGURE 23. COMPARISON OF IN VIVO TRANSDUCTION EFFICIENCY OF RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) RECOVERED FROM CP, SN, OR TC PROCESSES.. ................................ FIGURE 24: IN VITRO AND IN VIVO ANALYSES OF MSEAP ACTIVITY. .......................................................... FIGURE 25. LOCALIZATION OF DNA IN ISOPYCNIC IODIXANOL GRADIENT AFTER ULTRACENTRIFUGATION. ...................................................................................................................... FIGURE 26. TITRATION COMPARISON OF AAV VECTORS PURIFIED WITH OR WITHOUT ADDITION OF DNASE DURING THE CLARIFICATION STEP. ....................................................................................... FIGURE 27. SCHEMA COMPARING THE PREVIOUS PURIFICATION METHOD WITH THE NEW METHOD. .. FIGURE 28. COMPARATIVE CHARACTERISTICS OF RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) PURIFIED WITH CLASSICAL OR NEW METHOD, AND SPECIFICITIES OF THE NEW IODIXANOL DENSITY GRADIENT ON RAAV PARTICLES. ....................................................................................... FIGURE 29. QUANTITATIVE AND QUALITATIVE ANALYSES OF PILOT-SCALE PRODUCTIONS OF RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV)2/9 VECTORS PROCESSED BY THE NEW METHOD, AND EVALUATION OF THEIR IN VIVO PERFORMANCES. .................................................... FIGURE 30. SCHEMATIC REPRESENTATION OF THE RAW MATERIAL AND UPSTREAM PROCESSES USED FOR THE PRODUCTION OF RECOMBINANT AAV BY BACULOVIRUS EXPRESSION VECTOR SYSTEM. ................................................................................................................................................. FIGURE 31. IN VITRO TRANSDUCTION EFFICACY OF AAV2/9-GFP VECTORS PROCESSED BY CLASSICAL OR NEW PURIFICATION METHOD. ....................................................................................................... FIGURE 32. IN VIVO EFFICACY OF INTRAVENOUS INJECTION OF RAAV2/9-MSEAP PRODUCED FOLLOWING THE NEW VIRAL VECTOR PROCESS. ..............................................................................

  USED AAV VECTORS .... TABLE 2. TITER COMPARISON BETWEEN CP, SN AND TC PROCESSES (N=4).. ........................................... TABLE 3. DETAILED IN VITRO RESULTS OF MSEAP CONCENTRATION IN SUPERNATANT OF HEK293T CELLS TRANSDUCED WITH CP, SN OR TC DERIVED AAV VECTORS AT A MOI OF 10 5 . ................. TABLE 4. DETAILED RESULTS OF MSEAP CONCENTRATION (NG/ML) IN BLOOD OF INTRAMUSCULARLY INJECTED MICE. ...................................................................................................................................... TABLE 5. DETAILED COMPARISON BETWEEN CLASSICAL AND NEW AAV DOWNSTREAM PROCESSES. . TABLE 6. TITERS AND YIELDS ANALYZED FROM RAAV PRODUCTIONS PERFORMED AT 100 ML, 400 ML ET 5 LITERS.. ..........................................................................................................................................

  idea of gene therapy: a biological history of 20th century .................................. I.2 The natural gene transfers: the "horizontal" point of view ....................................... I.2.1 The "horizontal" gene transfer: generalities ........................................................................ I.2.2 Some natural examples of HGT .............................................................................................. a. HGT in prokaryotes .................................................................................................................. b. HGT in eukaryotes ................................................................................................................... I.3 Gene therapy: clinical application of the gene transfer strategies ............................ I.4 The artificial gene transfers: the science of vectorology ............................................. I.4.1 The "transgene" concept and the molecular cloning .......................................................... I.4.2 Gene transfer vectors: generalities ......................................................................................... I.4.3 Non-viral vectors: types and mode of action ........................................................................ a. Delivery methods of non-viral tools ...................................................................................... b. Delivery of naked DNA ........................................................................................................... c. Delivery of DNA by calcium phosphate precipitation ....................................................... d. Delivery of DNA by cationic lipids, liposomes and lipoplexes ....................................... e. Delivery of DNA by polymersomes ...................................................................................... I.4.4 Viral vectors: types and mode of action ................................................................................ a. Generalities ................................................................................................................................ b. Delivery of transgenes by Herpes simplex virus vectors .................................................. c. Delivery of transgenes by Retrovirus/Lentivirus vectors ................................................... d. Delivery of transgenes by Adenovirus vectors ................................................................... e. Delivery of transgenes by adeno-associated virus vectors ................................................

Figure 8 .

 8 Figure 8. Structure of AAV genome with the organization of the rep and cap genes and the resulting proteins. P5 and P19 are the promoters coding the four isoforms of the rep genes. P40 represents the promoter coding the three VP capsid proteins (VP1, 2 and 3). The VP1/VP2/VP3 ratio (1:1:10) is shown (right bottom) through a western blot analysis.

Figure 10 .Figure 11 .

 1011 Figure 10. Transposition process to produce rBAC and BIIC. The first step is the production of the raw material: recombinant baculovirus genomes (pBAC) are constructed, one harboring the transgene cloned between two ITR sites and the other encoding for the AAV rep/cap genes. The production of recombinant baculoviruses (rBAC) is performed by transfection of the Sf9 insect cell line with the pBAC. (Image from Invitrogen: The Invitrogen™ BaculoDirect™ Baculovirus Expression System.)

Figure 15 .

 15 Figure 15. Exon-skipping strategy. Taking the example of the mouse model mdx, a point mutation in the exon 23 leads to a premature stop codon and the production of a truncated non-

  ................................................................................................................ I. Gene vector processes: improvement of production methods for AAV vectors. ......... II. Gene vector application: gene editing for DMD ........................................................... MATERIAL AND METHODS .................................................................................... I. Methodology: Article 1: Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors (consult pages 89-93 for detailed methodology). ............................................................. II. Methodology: Article 2: Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system(consult pages 114-119 for detailed methodology). .................................................................................. III. Gene vector application: gene editing for DMD (consult pages 126-131 for detailed methodology) ..................................................................................................................... OBJECTIVES I. Gene vector processes: improvement of production methods for AAV vectors.

  However, both methods lead to the loss of a great amount of vectors left in the nonextracted fraction. My aim was to develop a new purification method in which viral vectors from the whole culture (cell pellet and supernatant) could be extracted at the same time, allowing the increase of the yields. These results were published in the journal Molecular Therapy Methods & Clinical Development: Dias Florencio, G., Precigout, G., Beley, C., Buclez, P.-O., Garcia, L., and Benchaouir, R. (2015). Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adenoassociated virus vectors. Mol. Ther. -Methods Clin. Dev. 2, 15024.

  These results were published in the journal Molecular Therapy Methods & Clinical Development: Buclez, P.-O., Dias Florencio, G., Relizani, K., Beley, C., Garcia, L., and Benchaouir, R. (2016). Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system. Mol. Ther. -Methods Clin. Dev. 3, 16035.

:

  Article 1: Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors (consult pages 89-93 for detailed methodology).

  In vivo experiments: C57BL/10 adult mice (3 animals/group) were injected with equal doses of AAV2/9-mSEAP derived from TC, CP and SN cultures. Two doses were tested: A high dose of 5.0 × 10 10 vg and suboptimal dose 1.0 × 10 10 vg were respectively injected intramuscularly in left and right tibialis anterior (TA) muscles of each mouse. Blood was drawn weekly for four weeks in order to measure mSEAP expression in the serum. After four weeks', animals were sacrificed and both TAs from each animal harvested. mSEAP staining was performed in muscle slides following the same protocol as in vitro experiments.II. Methodology: Article 2: Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system(consult pages 114-119 for detailed methodology).

  : gene editing for DMD (consult pages 126-131 for detailed methodology) Construction and rAAV production In order to induce homologous recombination two constructions were made. The first plasmid, named crmdx, carries the sgRNA and the Cas9 derived from Staphylococcus aureus. The sgRNA is 20 nucleotides long (sequence: GUAACAAAAUGGCUU-CAACUAU) and matches close to a Protospacer Adjacent Pattern (PAM) sequence located next to the mdx mouse mutation in exon 23. The second plasmid (crmdxΔ760), carries a HDR template harboring the correct wild-type dystrophin sequence that should be recombined into the genomic DNA. This 760bp template, extends 380bp to each side of the mutated nucleotide (T mutated/C wildtype) with the aim to replace the mdx mutation. Recombinant AAV9 were produced by tri-transfection following le protocol described in Dias-Florencio et al., 2015.

Figure 21 .

 21 Figure 21. Comparison of vector yields and purity between CP, SN, and TC processes. (a) Schematic representation of plasmid constructions used for recombinant adeno-associated virus (rAAV) production. The pAAV2/9 plasmid (upper part) encodes for the rep and cap genes. Regulatory and structural AAV proteins are controlled by their natural P5 (Rep78/68), P19 (Rep52/40) and P40 (VP1, 2, and 3) promoters. The cap genes encode for serotype 9 capsid. The helper adenoviral plasmid (middle part) encodes for helper functions. The pAAV-mSEAP cis-plasmid (lower part) encodes for the mSEAP reporter gene under the control of CMV promoter (Pcmv) and terminated by a non-constitutive polyadenylation site (pA). Transgene construction is cloned between two inverted terminal repeats

Figure 22 .

 22 Figure 22. Comparison of in vitro transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Light microscopy pictures of mSEAPpositive HEK293T cells 48h post-transduction. Compared to non-transduced cells (top left), rAAV originating from TC process (bottom right) are able to efficiently transduce permissive cells and express the reporter gene as well as viral vectors recovered from the classical CP or SN processes (top right and bottom left respectively). Black points correspond to mSEAP activity revelation into transduced cells (see Materials and Methods for staining protocol). Scale bar = 40 µm. (b) Quantification of mSEAP activity in supernatant harvested from wells of transduced HEK293T cells. Despite the relative 2.5-fold increase of mSEAP concentration between TC and SN rAAV transduction results, this difference is still significant (with P < 0.01). Refer to Supplementary Table3for more details.

Figure

  Figure 23. Comparison of in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) recovered from CP, SN, or TC processes. (a) Schematic representation of the in vivo experimental protocol. C57Bl/10 adult mice were intramuscularly injected (Tibialis anterior (TA)) with the same suboptimal doses (10 10 vg/TA) of CP, SN, or TC-derived rAAV, and blood is drawn weekly for one month before animal sacrifice and TA muscles recovery. Refer to Materials and Methods for detailed in vivo protocol. (b) The mSEAP revelation on TA muscle cryosections harvested 4 weeks post-transduction. Compared to PBS-injected muscle (bottom right), TCderived vectors offer widespread TA mSEAP staining (bottom left) at level visually comparable to that of CP or SN vectors (top left and right respectively). Larger sections views are shown at bottom right of each TA muscle cryosections. Scale bar = 2 mm. (c) For more quantitative monitoring, in vivo mSEAP concentration is evaluated in blood-derived sera. From the second week postinjection, the mSEAP serum concentration average appears systematically higher in mice injected with TC-derived rAAV than with injections performed from CP-or SN-derived vectors. A stabilization of the mSEAP concentration is observed from the third week postinjection. (b) and (c) are obtained from mice injected with suboptimal doses of rAAV. Refer to Supplementary Table4and Supplementary Figure24bfor more detailed table and pictures from TA muscles injected at higher rAAV doses.

  5 g/l of D-Glucose (Life Technologies, France), 10% of fetal bovine serum (Biowest, France) and 1% of Penicillin-Streptomycin solution (Life Technologies). Three days postseeding, HEK293T cells are tri-transfected by mixing 1,125 µl of 10 mmol/l polyethylenimine (Sigma-Aldrich, France) with 265 µg of pAdDeltaF6 helper plasmid, 105 µg of pAAV2/9 packaging plasmid (both from Penn Vector Core, University of Pennsylvania, USA), and 130 µg of pAAV-mSEAP cis-plasmid, all (both polyethylenimine and plasmids) previously diluted in 10 ml of 150 mmol/l NaCl solution. Transfection is performed in Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum and 1% Penicillin-Streptomycin solution.

Figure 28 .

 28 Figure 28. Comparative characteristics of recombinant adeno-associated virus (rAAV) purified with classical or new method, and specificities of the new iodixanol density gradient on rAAV particles. (a) Quantification by quantitative polymerase chain reaction of the total vg extracted from 100 ml productions purified by conventional or new downstream processes (n = 3). No significant difference was observed between the two methods. (b) Analysis of the quality of final

Figure 29 .

 29 Figure 29. Quantitative and qualitative analyses of pilot-scale productions of recombinant adenoassociated virus (rAAV)2/9 vectors processed by the new method, and evaluation of their in vivo performances. (a) Comparative quantification of total viral genomes extracted from 100 ml, 400 ml and 5l productions (n = 3). Linear productivity was observed between the different volumes of productions.The 100 and 400 ml suspensions were produced in Erlenmeyer flasks whereas the 5l were done in a bioreactor system. Supplementary details on titers and yields are available in Supplementary Table6. (b) The quality of the final rAAV product recovered from 5l production was determined by silver staining of 5 × 10 10 vg loaded on SDS-PAGE. The same high-quality vectors than previously described for research-scale productions were obtained (see Figure28b). The VP ratio was also maintained. (c) Analysis of mSEAP concentration in blood of C57BL10 mice intramuscularly injected with rAAV2/9-mSEAP vectors (n = 3). Continuous increase in levels of mSEAP was observed within 4 weeks after injection. The lower curve represents the levels of mSEAP in control mice injected with saline solution. (d) Histological staining of PBS (left panel) and rAAV2/9-mSEAP (right panel) injected muscle (Tibialis anterior). Serial sections were used for hematoxylin-eosin staining (upper sections) and for mSEAP enzymatic staining. Microscopic enlargement of the tissue sections is shown in the bottom. Saturating mSEAP expression was observed in rAAV-injected muscles. (e) Histological staining of different muscle tissues (TA, heart and diaphragm) originating from C57BL10 mice intravenously (IV) injected with PBS (left panel) or rAAV2/9-mSEAP vectors (right panel). As observed in (d), high mSEAP expression was also observed in different tissues, demonstrating the efficacy of rAAV vectors processed with our new purification method to perform whole body treatment. Additional results on IV efficiency of vectors are shown in Figure 32.

  Figure 29. Quantitative and qualitative analyses of pilot-scale productions of recombinant adenoassociated virus (rAAV)2/9 vectors processed by the new method, and evaluation of their in vivo performances. (a) Comparative quantification of total viral genomes extracted from 100 ml, 400 ml and 5l productions (n = 3). Linear productivity was observed between the different volumes of productions.The 100 and 400 ml suspensions were produced in Erlenmeyer flasks whereas the 5l were done in a bioreactor system. Supplementary details on titers and yields are available in Supplementary Table6. (b) The quality of the final rAAV product recovered from 5l production was determined by silver staining of 5 × 10 10 vg loaded on SDS-PAGE. The same high-quality vectors than previously described for research-scale productions were obtained (see Figure28b). The VP ratio was also maintained. (c) Analysis of mSEAP concentration in blood of C57BL10 mice intramuscularly injected with rAAV2/9-mSEAP vectors (n = 3). Continuous increase in levels of mSEAP was observed within 4 weeks after injection. The lower curve represents the levels of mSEAP in control mice injected with saline solution. (d) Histological staining of PBS (left panel) and rAAV2/9-mSEAP (right panel) injected muscle (Tibialis anterior). Serial sections were used for hematoxylin-eosin staining (upper sections) and for mSEAP enzymatic staining. Microscopic enlargement of the tissue sections is shown in the bottom. Saturating mSEAP expression was observed in rAAV-injected muscles. (e) Histological staining of different muscle tissues (TA, heart and diaphragm) originating from C57BL10 mice intravenously (IV) injected with PBS (left panel) or rAAV2/9-mSEAP vectors (right panel). As observed in (d), high mSEAP expression was also observed in different tissues, demonstrating the efficacy of rAAV vectors processed with our new purification method to perform whole body treatment. Additional results on IV efficiency of vectors are shown in Figure 32.

Figure 30 .

 30 Figure 30. Schematic representation of the raw material and upstream processes used for the production of recombinant AAV by baculovirus expression vector system. The illustrated steps precede those depicted in figure 27. Firstly, two recombinant baculovirus genomes (pBAC) were constructed, one harboring the AAV genome (GFP or mSEAP cassette cloned between two ITR sites)

4 . 5 . 6 .

 456 figure 36 compares the ESE profile of the wild type and the selected modified PAM sequence. This modification should predictably conserve an optimal ESE profile compared to the wild type exon 23 profile (Figure36).

  distinctive treatments described above. The gain of time obtained by the treatment of the cell culture by the Triton X-100 is advantageously amplified by the simultaneous addition of nuclease (RNAse A) enabling, during the same step, a significant reduction of the medium viscosity. The viscosity is a limiting parameter for subsequent purification steps. It is created by the release of nucleic acids from dead cells. As quantified by ourselves (data not shown), the principal nucleic acids responsible for viscosity are the RNA (mostly ribosomal RNA). Both, the detergent and nuclease treatments greatly facilitate the last step of clarification consisting of filtering the lysate through membranes with a pore diameter equal to 0.22 µm (sterilizing filtration).

I. 6 .

 6 Brain targeting with rAAV vectors. The blood brain barrier is a solid defense and not usually permissive to brain delivery of drugs and vectors injected into the bloodstream. For this reason, brain targeting is mostly subjected to invasive techniques that aims local delivery into the cerebrospinal fluid or directly into the brain parenchyma. Recently developments are showing that a few serotypes of rAAV are indeed capable to cross the blood brain barrier. Following the delivery of high titers of rAAV2/9-mSEAP by retroorbital intravenous injections, we were able to detect mSEAP expression in the brain. Even though the targeting is not yet robust, this can open the field for the treatment of some neuronal diseases, suppressing the need for invasive methods of local injections. This method can yet be coupled with a cell-specific (either neuronal, astrocyte or microglial) promoter to inhibit expression in other tissues in the periphery of the body. One question remains unanswered though: what cell type is AAV 2/9 targeting in the brain? mSEAP expression was identified in several brain areas as shown in the figure 34. Mainly round small purple dots scatted throughout several areas were found.

•

  Seed 15cm petri dishes with 17.10 6 HEK293T cells in DMEM medium supplemented with 4,5 g/l glucose, + GlutaMAX -pyruvate, 10% SVF, 1% penicillin/streptomycin. One petri dish per Hyperflask (HF). 24h after dishes should be at 80% confluence (35x10 6 cells)•Detach cells and re-suspend in 200mL in same composition medium;• Transfer cells to the HF equally divided in all layers, and fill the HF with culture medium qsp 560 mL.2.TRANSFECTION: 3 days after seeding, transfect cell culture to HFs:One HF = 500µg of DNA in total. Calculate according to the table below: In a 50mL tube add the three plasmids according to the volume previously calculated and add NaCl 150mM qsp 10mL; • In a second 50mL tube add 1,125mL of PEI 10mM and NaCl 150mM qsp 10mL * PEI 10mM volume (in µl) = 2,25 x total DNA mass (in µg): 2,25 x 500 = 1125µl • Complex DNA and PEI under shaking drop by drop;

• A 0 .

 0 22 µm polyvinylidene difluoride membrane of 0.1 or 0.65 cm 2 (Millipore, Villepinte, France) was used for sterilizing filtration of the diafiltrated solution issue from research-scale or pilot-scale productions respectively. PT03. Titration of AAV vectors 1. Sample treatment • For 5µL of purified virus add: 1 µl DNase 10 u/µl + 45 µl DNase buffer. of Proteinase K 20 mg/ml + 195 µl DNase buffer. Incubate 1h à 50°C * Proteinase K buffer: 0.5% SDS / 10mM EDTA filtered with 0.45µm filter 2. Q-PCR TaqMan • 10 µl of the treated sample: dilute serially to obtain 10 -2 to 10 -7 of AAV DNA dilutions.

  -silver staining of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) -Loading 5 × 10 10 vg on 4-12% NuPage Bis-Tris gels (Thermo Fisher Scientific) -Run under reducing conditions.

  ) -Magnesium Chloride Hexahydrate (99% ACS reagent) MM 203.3 CaCl2 (50 mM) -Calcium chloride dehydrate MM 147.01 Preparations before starting:-supplemented HBSS, termed HBSS + : 1% P/S (add 5mL in bottle) and 0,2% BSA (10 ml of 10% BSA 1g in 10 ml filtered through 0* 25 = 1.84 g dissolved in 25 ml of sterile water -aliquoted Collagenase 100 µg/µL: for 500 mg add 5mL of sterile PBS 1x, aliquot in 500 µl -aliquoted DNAse I 10 mg/ml: for 100 mg add 1 mL, aliquot in 50 µl (Careful, do not vortex DNase) Procedure -Weigh needed amount of Dispase II and place in 15 ml falcon tube -Sacrifice mouse by cervical dislocation, spray the entire mouse with 70% ethanol, remove skin from the foot to the hip to expose underlying muscles, use sterile dissection tools to dissect both hindlimbs (Quadriceps, Tibialis Anterior, EDL, Gastrocnemius, Soleus, Plantaris, Gluteus, etc.), take off as much fat and connective tissue as possible (white tissues), place muscles on a 6 cm Petri dish containing HBSS + and placed on ice.Note: the better and faster you dissect the higher will be the initial myoblast yield -Go under the hood -Thaw collagenase -Still on ice, take new 6 cm Petri dish, in which you transfer the dissected muscles after inspecting and removing any remaining fat and connective tissue -Mince with curved scissors all the muscles until you obtain smooth pulp (do not leave chunks, they will not be digested thoroughly) -Split the pulp in two and transfer minced muscles in two 15 mL Falcon (for better digestion), add up to 10 mL of HBSS + , mix well -centrifuge 1300 rpm x 1 min 4°C, remove all the supernatant (to remove residual fat) -Add freshly prepared digestion mix (8 ml per mouse which will be divided in 4 ml in each of the two falcons), filtered through 45 µm (dispase is always dissolved freshly):Mother final solution in 8 ml Quantity needed Dispase II powder 0.9 U/mg 2,4 U/mL 21,3 mg HBSS + Qsp 8 Ml Collagenase A (in PBS) 100 µg/µL 2 µg/mL 160 37°C for 1h30-2h, vortex every 10 min -Add up to 15 mL HBSS + in each falcon -Pass both 15 mL through 100 µm cell strainer in the same 50 mL falcon -Centrifuge 1300 rpm x 5 min 4°C, discard supernatant, resuspend in 30 ml HBSS + Note: when resuspending the pellet of cells during washes: first in 1 mL and then add the rest of the volume -Pass through 70 µm cell strainer in a 50 ml falcon -Centrifuge 1300 rpm x 5 min 4°C, discard supernatant, resuspend in 30 ml HBSS + -Pass through 40 µm cell strainer in a 50 ml falcon -Centrifuge 1300 rpm x 5 min 4°C, discard supernatant Peprotech 450-33B, stock at 4000x, 0.01 mg/ml) Preparations before starting:-aliquot bFGF 10 µg/ml (4000x): for 50 µg add 625 µl, aliquot in 50 through 45 µm) Procedure -Resuspend pellet with 20 ml of growth media

  Vector genome titration by Q-PCR ................................................................................................. Silver staining of SDS-PAGE .......................................................................................................... In vitro assay ....................................................................................................................................... In vivo assay ........................................................................................................................................ Statistical analyses .............................................................................................................................

PART 2 II. Recombinant Adeno-Associated Virus: an attractive viral vector for gene therapy .. II.1 Wild type Adeno-associated viruses

  .............................................................................

II.1.

1 Structure, genome, serotypes and tropism

  ..........................................................................

II.1.2 Modes of infection: lytic versus lysogenic cycle ................................................................ II.1.3 Immunity and seroprevalence ............................................................................................... II.2 Recombinant AAVs .......................................................................................................... II.2.1 Research scale processes: the tri-transfection method ...................................................... II.2.2 Industrial scale processes: Baculovirus / insect cell system .............................................

rBac-Rep/Cap rBac-TG rBAC BIIC Co-culture BIIC -SF9 (ratio V BIIC /V Sf9 = 10 -4 ) rAAV (Baculovirus-Infected Insect Cells) Avantages : .cryoconservation

	INTRODUCTION
	PART 3
	Once the two BIIC are generated à long terme des rBAC (« protégés » par les BIIC)
	(one carrying the transgene and the other carrying rep/cap genes), the upstream process is initiated by
	co-cultivation of these two BIIC with native Sf9 cells in Erlenmeyer systems (for research scale
	projects) or bioreactor systems (for pilot scale projects). Production is harvest and then purified.

III. rAAV: viral vectors for gene therapy of Duchenne Muscular Dystrophy (DMD) ... III.1 DMD: general presentation of the genetic disorder

  .................................................

III.

1.1 DMD physiopathology

  .......................................................................................................... a. Clinical features ......................................................................................................................... b. The dystrophin gene ................................................................................................................ c

. The dystrophin protein and dystrophin-associated protein complex (DAPC)

  ..............

III.1.2 Genetic and differences between Becker muscular dystrophy and Duchenne muscular dystrophy

  .............................................................................................................................................

III.1.3 Animal models

  ........................................................................................................................ a. mdx mouse ..................................................................................................................................

b. Double knockout dystrophin/utrophin (dKO or mdx/utrn -/-)

  ........................................... c. mdx52 ...........................................................................................................................................

d. Dmd-mutated rats

  ......................................................................................................................

e. Canine model (GRMD)

  ............................................................................................................ f. Pig model ..................................................................................................................................... g. Other models ..............................................................................................................................

III.2 Therapeutic approaches for Duchenne muscular dystrophy using recombinant Adeno-associated virus

  ...........................................................................................................

III.

2.1 Mini-& micro-dystrophin strategies

  ..................................................................................

III.2.2 Exon-skipping approach (U7snRNA)

  .................................................................................

III.2.3 Genome editing

  ...................................................................................................................... a. Generalities ................................................................................................................................ b. Meganucleases ...........................................................................................................................

c. Zinc-Finger Nucleases .

  .............................................................................................................

d. Transcription activator-like effector nucleases (TALENS) .

  ..............................................

e. CRISPR-Cas9 .

  .............................................................................................................................

III. rAAV: viral vectors for gene therapy of Duchenne Muscular Dystrophy (DMD)

	Therapeutic strategies based on gene transfer are starting to give promising
	clinical results and open the way to cure many diseases. In this context, Duchenne
	Muscular Dystrophy (DMD) is commonly considered as model case for therapeutic
	approaches for genetic disorders. One reason for this attention is related to the
	nature of the pathophysiology itself, that affects the whole body, challenging
	researchers and physicians to fight against this deadly disease. Indeed, DMD is a
	hereditary disease that leads to progressive degeneration of the entire musculature.
	Besides the loss of skeletal muscle and therefore the mobility, the occurrence of
	cardiac hypertrophy, accompanied by degeneration of diaphragmatic muscles,
	lead to serious heart and respiratory failure, leading causes of death in DMD
	patients.
	III.1

DMD: general presentation of the genetic disorder III.1.1 DMD physiopathology a. Clinical features

	Duchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disease,

ACKNOWLEDGEMENTS

This thesis is the result of 4 years of research, sweat and tears. In this preamble, I would like to express my appreciation to those with whom I have been

RESULTS

Downstream process

As described in this paper, we investigated new purification process and compared it to previously published methods (see Figure 20a). The first protocol uses mechanical cell detachment by manually shaking the Hyperflask. Cell suspension (560 ml) is collected and 440 ml of 1× PBS is used to wash the Hyperflask. The 1L cell suspension is slowly centrifuged 30 minutes at 500×g to avoid physical cell lysis.

Supernatant (SN) is cautiously harvested in new vessel, and cell pellet (CP) is pretreated by pipeting with 18 ml of lysis buffer (0.15 M NaCl, 50 mmol/l Tris HCl pH 8.5). CP prelysate is then processed through four cycles of freezing/thawing each consisting 30 minutes incubation in absolute ethanol cooled in dry ice followed by a 15-minute incubation in a 37 °C water bath. Nuclease treatment is performed by using 50 U/ml of benzonase (VWR, France) for 60 minutes at 37 °C. Clarification process is ended by centrifugation of cell lysate at 3,700×g for 20 minutes followed by filtration through a 0.45 µm polyethersulfone membrane syringe filter unit of 33 mm diameter (Millipore, France). The 1 l SN is treated 2 hours with 5 µg/ml of RNase A (Sigma-Aldrich, France) at 37 °C under strong magnetic shaking. SN is then filtered through a 0.8-0.45 µm sartoscale 13 cm 2 double layer polyethersulfone membrane filter (Sartorius-Stedim, Germany).

The third protocol we investigated here drastically simplifies previous described methods. The 560 ml supernatant of the original Hyperflask is slowly collected in a vessel and treated with 3 ml of Triton X-100 nonionic detergent (Sigma-Aldrich), corresponding to 0.5% final concentration and 5 µg/ml of RNAse A (Sigma-Aldrich).

After rapid homogenization, the supernatant is transfered to the original Hyperflask and incubated for 1 hour at 37 °C under vertical rotating shaking performed at 150 rpm. TC suspension is collected in a vessel and 440 ml of 1× PBS is used to wash the Hyperflask. The washing suspension is added to the first 560 ml and the 1L volume is centrifuged at 3,700×g for 30 minutes. The supernatant derived from TC production is then filtered through the same 0.8-0.45 µm sartoscale filter than previously described for the SN filtration.

While CP filtrate is consecutively purified by isopycnic ultracentrifugation (see description below), SN and TC filtrates (1L each) are first concentrated by TFF. The TFF was processed by using the automated Kros Flow Research IIi TFF system (Spectrum Laboratories, France) through a 115 cm 2 polyethersulfone membrane hollow fiber unit with 500 kDa molecular weight cut off. SN and TC filtrates are concentrated 30 times, corresponding to a final volume of 35 ml.

The three methods are ended by an isopycnic ultracentrifugation derived from previously described protocol [START_REF] Zolotukhin | Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[END_REF]. Briefly, 17 ml of viral suspension is deposited through a glass Pasteur pipette in the bottom of 25 × 89 mm Quick-Seal Polyallomer dome-top tube (Beckman Coulter, France). This suspension is raised up by successive addition of 9 ml of 15% iodixanol (from 60% stock solution;

Sigma-Aldrich) at 1M NaCl, 5 ml of 25% iodixanol, 5 ml of 40% iodixanol, and 3 ml of 60% iodixanol. Iodixanol solutions are all diluted in 1× PBS-MK buffer (1M NaCl, 1 mmol/l MgCl2, 2.5 mmol/l KCl, 1× PBS). Tubes are heat-sealed with the Tube topper system (Beckman Coulter), placed in Ti70 rotor, and ultracentrifuged at 500,000×g

(70,000 rpm) for 70 minutes in an Optima L100XP ultracentrifuge (Beckman Coulter).

The Fraction Recovery system (Beckman Coulter) is used to puncture the tube from the bottom side: the six first milliliters, containing viral particles, are collected and diluted twice in 1× PBS before their diafiltration through centrifugal concentration and filtration unit Amicon Ultra-15 (Millipore, France) according to the manufacturer instructions. The diafiltration is performed three times leading to a 450 times dilution factor of the original collected solution. This step is ended by a 6 to 10 times concentration step (0.9 to 1.8 ml final volume).

Vector genome titration by Q-PCR 5 µl of purified vector is treated 30 minutes at 37 °C with 5 µl of DNAse I at 2U/µl (Ambion) diluted in 40 µl of DNAse buffer (13 mmol/l TRIS pH7.5, 0.12 mmol/l CaCl2, 5 mmol/l MgCl2). Five microliters of Proteinase K at 20 mg/ml (Ambion) diluted in 195 µl of Proteinase K buffer (0.5% SDS, 10 mmol/l EDTA) are added to previous mixture, Diafiltration was done through centrifugal concentration and filtration unit Amicon Ultra-15 (Millipore, Guyancourt, France) according to the manufacturer instructions.

RESULTS II

The diafiltration was performed three times leading to a 450 times dilution factor of the original collected solution. A 0.22 µm polyvinylidene difluoride membrane of 0.1 or 0.65 cm 2 (Millipore, Villepinte, France) was used for sterilizing filtration of the diafiltrated solution issue from research-scale or pilot-scale productions respectively.

AAV genome titration by Q-PCR

AAV genomes were titered as previously described [START_REF] Dias Florencio | Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors[END_REF].

Briefly, 5 µl of purified vector was treated 30 minutes at 37 °C with 5 µl of DNAse I at 2U/µl (Thermo Fisher Scientific). Five microliters of Proteinase K at 20 mg/ml (Thermo Fisher Scientific) were added to previous mixture, followed by 1 hour incubation at 56 °C. Ten microliters of pretreated solution were serially diluted to obtain 10 -2 to 10 -

Determination of AAV purity by silver staining of SDS-PAGE

Purity of recombinant AAV vectors was assessed on SDS-PAGE by loading 5 × 10 10 vg on 4-12% NuPage Bis-Tris gels (Thermo Fisher Scientific) run under reducing conditions. After migration, proteins and nucleic acid content was revealed by using Silver Staining Plus protocol (Bio-Rad Laboratories) following manufacturer instructions. The gel was deposited on white tray for picture acquisition by Gel Doc EZ system and analyzed by the Image Lab software (Bio-Rad). High hopes for the use of these types of vectors in therapy is now materializing by the first marketing approval granted to a rAAV based product by the European Union in 2012. The strict conditions imposed by European authorities aims to guarantee the safety of patients receiving the proposed treatments. This is a first step proving the safety of this technology, when well designed and rationally engineered. Together with the increasing number of clinical trials using this technology, treatment for many previously incurable diseases may emerge in the following years.

RESULTS III

II. CRISPR-Cas9: evaluation of a homologous recombination approach for DMD

Many studies attempt to transfer mini-and micro-dystrophin genes or used U7snRNA to induce exon-skipping and recover a truncated protein. This idea came from observations of the BMD mutations. Indeed, BMD patients present milder phenotype with almost normal life expectancy, contrary to the progressive and aggressive DMD phenotype. However, the transfer of short isoforms will maintain a dystrophic scenario because they are not as stable as the full-length form. In addition, these modifications either by insertion of an episomal gene form or RNA modulation are not permanent, and with loss of transgene, the muscles will again deteriorate. Moreover, when coupled with viral vector delivery, a second injection of these technologies is not viable because, after the first injection, the body will immediately start to produce antibodies against the vector. Gene editing offers the possibility of inducing permanent modification at the genomic DNA level. This is a major advantage over previous technologies. Briefly, as already described, with gene editing tools such as ZFN, TALENs and CRISPR-Cas9, the action of endonucleases will introduce a double strand break in the DNA. This DSB can be repaired by one of two methods: NHEJ uses the cell machinery to randomly repair the DSB, and this correction will introduce short DNA insertions or deletions. The other strategy, HDR, requires a template from where the cell machinery will copy the normal gene sequence. NHEJ-mediated gene correction offers several advantages: no need for a template, which will implicate in more efficient rate of correction, and NHEJ operates efficiently in all stages of the cell cycle and therefore

GENERAL SUMMARY OF THE CONCLUSIONS

The results presented here show new improvements in the biotechnological field of rAAV production both at research and pilot scale. The first method, for research scale, using a simple detergent treatment, increased the yields, reduce cost and production time, and surprisingly ameliorate the in vitro and in vivo transduction efficiency of the rAAV vectors. For the pilot method, simply by modifying the isopycnic iodixanol gradient, we were able to double the capacity for purification of AAV vectors. In addition, the reduction of the volume recovered following ultracentrifugation can indeed increase the quality of the final product by eliminating the empty AAV particles present in the upper fraction of the bottom iodixanol phase. The large biodistribution of the viral vector throughout the whole body gives us hope for an implementation of this type of processes for projects with a clinical focus.

Our preliminary results showed that is indeed possible to induce gene editing by homologous recombination in vivo using the CRISPR-Cas9 system in adult animals. For this to happen, however, the muscle must be in regeneration or • Discard supernatant -Or use for SN method.

• 4 cycles of freezing (dry ice + EtOH)/thawing(37°C) -always vortex in between

•

Possibility to conserve at -80°C before purification.

• 60 minutes' nuclease treatment (50 U/ml of benzonase) (VWR, France) at 37 °C

•

Centrifugation of cell lysate at 3,700×g for 20 minutes followed by filtration through a 0.45 µm polyethersulfone membrane syringe filter unit of 33 mm diameter (Millipore, France).

SN method

• Supernatant recovered from CP method is N is treated 2 hours with 5 µg/ml of RNase A (Sigma-Aldrich, France) at 37 °C under magnetic shaking.

• Supernatant is filtered through a 0.8-0.45 µm sartoscale 13 cm 2 double layer polyethersulfone membrane filter (Sartorius-Stedim, Germany).

• TFF is performed by an automated Kros Flow Research IIi TFF system (Spectrum Laboratories, France) using a 115 cm 2 polyethersulfone membrane hollow fiber unit with 500 kDa molecular weight cut off. Final volume is approximately 35mL.

TC method

•

The supernatant is transferred to a sterile bottle in order to homogenise the detergent (Triton X-100 3mL -0,5% final concentration) and RNase A (5 µg/ml -Sigma-Aldrich). The supernatant is transferred back to the HF for 1h incubation under mechanical shaking at 37°C.

• TC suspension is collected in a sterile bottle and 440 ml of 1× PBS is used to wash the Hyperflask. The washing suspension is added to the first 560 ml and the 1L final volume is centrifuged at 3,700×g for 30 minutes.

• Supernatant is filtered through a 0.8-0.45 µm sartoscale 13 cm 2 double layer polyethersulfone membrane filter (Sartorius-Stedim, Germany).

• TFF is performed by an automated Kros Flow Research IIi TFF system (Spectrum Laboratories, France) using a 115 cm 2 polyethersulfone membrane hollow fiber unit with 500 kDa molecular weight cut off. Final volume is approximately 35mL. promoter, and were cloned in pFastBac 1 plasmid to produce the pFBD-GFP or pFBD-mSEAP donor plasmids respectively.

Generation of rBAC

•

The pFBD-Rep2/Cap9opt, pFBD-GFP, and pFBD-mSEAP were each transformed in DH10Bac competent Escherichia coli cells to produce, by transposition mechanism, the pBAC-Rep2/Cap9opt, pBAC-GFP, or pBAC-mSEAP recombinant baculoviral genomes respectively.

Generation of BIIC