David Klatzmann
email: david.klatzmann@upmc.fr

Pr David Klatzmann

A Sophism in Vectorology: Turning Harmful Defective Retroviral Vectors into Helpful Replication-Competent Retroviruses Against Cancer

Protagoras (as cited in Plato's Cratylus, 386a) ''Of all things the measure is Man, of the things that are, that they are, and of things that are not, that they are not.

I bumped into a smiling guy as we headed to the same spot, the far end of a large poster hall of the Third Annual American Society of Gene Therapy (ASGT) Meeting in 1999 in Washington DC. It was as if we had been banished to the remotest part of the hall, so as not to be seen. We unfolded and hung our posters. And we understood. Nori Kasahara and I were both presenting our results on the development of replication-competent retroviral (RCR) vectors for gene therapy (of cancer). And this was right at a time when everyone who wanted to use defective retroviral vector for a clinical trial knew they would be scrutinized by the regulatory agencies for the presence of RCR in their vectors' preparations. Any RCR and the preparation were trashed. And there were scientists seriously thinking of using RCRs for gene therapy! How is that possible?

For me, it all started because of the results of our clinical trials of suicide gene transfer for the treatment of patients with glioblastoma (GBM) [START_REF] Klatzmann | Gene therapy for glioblastoma in adult patients: safety and efficacy evaluation of an in situ injection of recombinant retroviruses producing cells carrying the thymidine kinase gene of the herpes simplex type 1 virus, to be followed with the administration of ganciclovir[END_REF] and melanoma. [START_REF] Klatzmann | Gene therapy for metastatic malignant melanoma: evaluation of tolerance to intratumoral injection of cells producing recombinant retroviruses carrying the herpes simplex virus type 1 thymidine kinase gene, to be followed by ganciclovir administration[END_REF] Preclinical results on the use of suicide genes to treat experimental cancer in rodents had been spectacular. [START_REF] Culver | In vivo gene transfer with retroviral vector producer cells for treatment of experimental brain tumors[END_REF][START_REF] Caruso | Regression of established macroscopic liver metastases after in situ transduction of a suicide gene[END_REF] The experimental system appeared to be loaded with fantastic properties. Killing cells was clearly an easier task than fixing them, as needed for the treatment of genetic diseases. There were no (less) issues on how much transgene expression was needed, and for how long. Killing cells with suicide genes, which needed the combined expression of the gene and the administration of a prodrug (ganciclovir [GCV] in our case), appeared to have robust safety features. Indeed, the retroviral infection required cell division and so did GCV's killing mode of action, together ensuring good specificity of the killing of tumor cells. And there were these fantastic bystander effects-local [START_REF] Gagandeep | Prodrugactivated gene therapy: involvement of an immunological component in the ''bystander effect[END_REF] and distant [START_REF] Kianmanesh | A ''distant'' bystander effect of suicide gene therapy: regression of nontransduced tumors together with a distant transduced tumor[END_REF][START_REF] Wei | Suicide gene therapy of chemically induced mam-mary tumor in rat: efficacy and distant bystander effect[END_REF] -which alleviated the need for very efficient gene transfer (or so we thought). So, after seeing these large liver tumors disappear in rats, [START_REF] Caruso | Regression of established macroscopic liver metastases after in situ transduction of a suicide gene[END_REF] I enthusiastically turned to the translation to clinical evaluation, which proved to be a bumpy road.

At the time, scientists (at least me) hardly knew what ''good manufacturing practices'' (GMP) meant. I rapidly learned two things about them: they are labor-intensive and expensive. So, we now had to look for additional funding. In France, at the time, there was no specific funding for translational research in gene therapy, only for experimental preclinical research. Requests for significant amounts of money just to produce a ''clean'' packaging cell line were received by grant committees with comments such as ''this is not science.'' To cut a long story short, after trying GMP production with the only two companies ''capable'' of producing retroviral vectors in Europe at the time, and after depressing failures, we had no choice but to do it ourselves-which meant launching, with my colleague and friend Jean-Loup Salzmann, a biotech company that we pompously called Ge ´nopoı ¨e ´tic (what a nice name!). Start-ups were another discovery, plus working with other types of people. Ge ´nopoı ¨e ´tic's first goal was to set up the GMP production of retroviral vectors, with the help of Rho ˆne-Poulenc Rorer (now Sanofi). Thanks to the talent of the late Andre ´Crespo, it worked, and in a timely fashion, we produced our first approved batches. Incidentally (and ironically), at the same time, we produced the first batches of retroviral vectors used for the first trial of gene therapy of severe combined immunodeficiency, [START_REF] Cavazzana-Calvo | Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease[END_REF] the very trial that raised concern about the safety of defective retroviral vectors.

With our vectors, we launched what were at the time the first two European cancer gene therapy clinical trials, based fully on local research, development, and production. This was at the time when Human Gene Therapy had started to publish clinical trials. [START_REF] Klatzmann | Gene therapy for glioblastoma in adult patients: safety and efficacy evaluation of an in situ injection of recombinant retroviruses producing cells carrying the thymidine kinase gene of the herpes simplex type 1 virus, to be followed with the administration of ganciclovir[END_REF][START_REF] Klatzmann | Gene therapy for metastatic malignant melanoma: evaluation of tolerance to intratumoral injection of cells producing recombinant retroviruses carrying the herpes simplex virus type 1 thymidine kinase gene, to be followed by ganciclovir administration[END_REF] These trials were the first to go through the regulatory committees that had just been formed at the time in France, under the direction of Jean-Hugues Trouvin. Having had to learn about, and then deal with, GMP production, we now had to learn about regulatory issues and in fact help to build with the committee what became the standards for the upcoming trials. We finally received regulatory clearance for our trials.

The treatment modality was to inject the packaging cells producing the defective retroviral particles that transduce suicide genes straight into the tumor. Compared to the injection of viral particles, we thought that injection of packaging cells would improve tumor cell transduction. GBM and melanoma were chosen for reasons that would take too long to explain here. They retrospectively have been worst rather than best choices. In these firstin-man trials, we had to treat advanced (euphemism) diseases. We could not treat all the multiple lesions of our melanoma patients, and although we saw remarkable regression of some injected tumors, the distant bystander effects were obviously insufficient to take care of the enormous disseminated tumor mass from which these patients suffered. [START_REF] Klatzmann | A Phase I/II dose-escalation study of herpes simplex virus type 1 thymidine kinase ''suicide'' gene therapy for metastatic melanoma[END_REF] For the GBM patients, the setting looked more favorable, with a tumor that often appeared to be well limited on magnetic resonance imaging (MRI). Patients had their tumor surgically debulked, and the cells were injected quite cautiously and meticulously all around the tumor margin. The atmosphere in the operating room was quite something, with an entire team of nurses and doctors having the impression that they were making history. We treated 12 patients, with ''mixed'' results. [START_REF] Klatzmann | A Phase I/II study of herpes simplex virus type 1 thymidine kinase ''suicide'' gene therapy for recurrent glioblastoma[END_REF] At 4 months after treatment, four patients had no recurrence, and later had an overall survival almost three times longer than patients with recurrence. One patient was still free of detectable recurrence, steroid free, and independent 2.8 years after treatment, which was quite unusual. So, the glass looked pretty empty to most of us. However, one of most striking observations for me came from the postmortem pathological studies. It is the tradition in my hospital that the brains of patients are macroscopically examined in the presence of the entire staff of doctors and students, in a ''Charcot style session.'' The entire brain is placed on a plate, and is then sliced. Having skipped neuropathology during my medical studies, the vision was quite disturbing. These were the brains of people I had been speaking with just a few days earlier-people who had placed in us their last hopes, and whom we had failed. However, these sessions revealed something that puzzled me greatly. Even on macroscopic slices, it was so obvious that the tumor was not the well-limited mass we saw on the MRI. There were migrating tumor cells everywhere, even in the contralateral hemisphere. I knew from the literature that GBM does not generate metastases but is disseminated locally. But to that extent? How naı ¨ve I had been to think that we could target these cells with our technique.

This could have been it: empty glass, failure, end of the program. But the enormous amount of work-of team work-put into this research and my optimistic nature made me see something in the glass. It was not even half-full, but something was there. The one long-term survivor at the time that we wrote our report later died from disseminated breast cancer, with still no indication of tumor recurrence on MRI. Postmortem analysis confirmed that there was no evidence of recurrence near the initial tumor localization or in any other area of the brain. The pathologists were amazed and agreed to conclude a case report with ''Such an evolution is unusual in the course of this disease and may suggest in this particular case a cure of the GBM.'' [START_REF] Vale ´ry | Long-term survival after gene therapy for a recurrent glioblastoma[END_REF] To write ''cure'' for GBM was quite dramatic.

What did this patient's story teach us? That maybe this treatment could work. What if we had injected her ''better''? What if the gene transfer had been more efficient? The optimistic lesson from this case was that it was all about the efficiency of gene transfer. Efficacy called for efficiency (of transfer). How can this efficiency be achieved? Then came this crazy idea: why work with defective vectors, which were dead after their single shot? Why not inject replicative vectors? Any locally transduced tumor cell could then produce more viruses, to infect more surrounding cells, and so on and so forth. Tempting, but what about safety? Well, these patients have a median survival of 8 months. Would they really care about possible leukemia that could potentially be controlled with chemotherapy and could be deadly in years? And any infected virus-producing cell would express the suicide gene, the potential leukemic cells also, making them targets for GCV. I turned this around and around in my mind, and realized that it would all be about efficacy in experimental models. If it was not that much more efficient in mice, that would be the end of the story. If it was, however, then it would be time to discuss safety with experts. And so we started. And it worked nicely in mice. We submitted an abstract to the ASGT and were rather disappointed not to be able to report our work in an oral presentation. Although many researchers had started long ago to make RCR for various purposes (reviewed by Dalba et al. [START_REF] Dalba | Beyond oncolytic virotherapy: replication-competent retrovirus vectors for selective and stable transduction of tumors[END_REF][START_REF] Dalba | Replication-competent vectors and empty viruslike particles: new retroviral vector designs for cancer gene therapy or vaccines[END_REF] ), obviously, RCRs for a clinically oriented development were low in the pecking order.

And so it was that we met with Nori. Being ''parked'' together likely helped build our collaboration and friendship. We could have been competitors (we had both applied for patents at almost the same time), but we collaborated. We published articles reporting the amazing efficacy of these vectors. [START_REF] Wang | Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors[END_REF][START_REF] Solly | Replicative retroviral vectors for cancer gene therapy[END_REF] We reported improvements in use, such as transduction through DNA gene transfer of replicationcompetent genome. And we did not overlook safety. Being half worried that the regulatory authorities would not allow the use of RCRs, I developed the concept of semi-replicative vectors. [START_REF] Trajcevski | Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors[END_REF] These are two complementary vectors that can produce only half of what is necessary for replication, gag/pol or env, plus a therapeutic transgene. They beautifully (yes, beautifully, this is how I saw it) spread in vitro and in vivo and ''never'' recombined to form fully replicative vectors. We also did some preclinical safety studies showing that RCRs could not be detected in the periphery. However, obtaining authorization for clinical evaluation appeared far away.

But then light emerged at the end of the tunnel. Nori met with Doug Jolly, one of the pioneers of suicide genes who well appreciated the concept. Soon, a company was formed (Tocagen) and, yes, successfully raised the big money needed to start the endeavor. That there were people willing to invest on RCRs at the time was quite amazing to me. It could not have happened here in France. Nori was involved, but the deal of these entrepreneurs with my university, which holds my patent on this work, never worked out. The preclinical work had been completed, and it was now time for clinical trials. No way could I do it on my own and compete with this company, so I gave up, regretting that I had been unable to bring this concept all the way to the clinic.

The vector was optimized and produced under GMP. The Food and Drug Administration authorized a first clinical trial, with no major difficulties, as far as I know! I doubt it would have been that easy here. Forty-five patients with gliomas were treated. [START_REF] Cloughesy | Phase 1 trial of vocimagene amiretrorepvec and 5fluorocytosine for recurrent high-grade glioma[END_REF] The treatment was well tolerated and improved survival, opening the way to a Phase 2/3 trial. But that's not all. Tocagen investigated whether it would be possible to administer the RCRs intravenously! Their preclinical data in rodents did indeed support this route of administration. The vector reached the tumor and efficiently spread within the tumor. [START_REF] Huang | Intravenous administration of retroviral replicating vector, Toca 511, demonstrates therapeutic efficacy in orthotopic immune-competent mouse glioma model[END_REF] Based on these results, a clinical trial investigating intravenous administration of suicide genes carrying RCRs has been approved and is now enrolling patients! At this early stage of development, it is of course difficult to predict how efficient these first-generation vectors will be. I am quite optimistic that they will show some efficacy and lead to the building of increasingly efficient second-and then third-generation RCRs.

What can be learned from this story? In my first scientific life, when I was working on human immunodeficiency virus, I was lucky to learn science in an incredibly diverse environment, where you had to be multifaceted, doing immunology and molecular biology while also working with sociologists, anthropologists, economists, journalists, politicians, lobbyists. That was quite an education. In my second scientific life, I still do not know whether I should consider myself lucky to have entered the field early. You also had to be multifaceted, but this time through learning about production, regulatory affairs, business, and more, all of which are important but somehow less fun, for me at least. At the time, translational gene therapy called for resilience, adaptability, hard work, and much more for return on investments (academic I mean) that were often low! Having now turned to protein-based biotherapy, [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF] I appreciate even more the handicap from which gene therapists suffer. I remember one of my post-docs from years ago. She was a bacteriologist. The bacteria were grown overnight, and it was almost always possible just to put them in the freezer to stop the process and have time off if there was an invitation for a party. Nothing we could afford. Anyhow, the technological aspects of gene therapy, the smartness that can be put into it, are still thrillingly interesting to me. So, I have not fully betrayed the field, because my now preferred protein, interleukin-2, 20 can be administered (advantageously?) by viral vectors! And we are exploring this avenue of research. [START_REF] Churlaud | Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer[END_REF][START_REF] Alves | Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer's disease mice[END_REF] Another lesson is that much (everything?) is about devoting enough time to good, deep, independent thinking. Think, think, think, think your own thoughts. And finally, it is also very much about people-those you work with and those you work for, the patients. My only wish now is that these RCRs, this generation, and the many improved generations to come will save many lives.

To conclude, let's not forget what Protagoras taught us: that there are always ways to ''make the worst case the better'' or, to put it another way, to transform a weakness into a strength.

ACKNOWLEDGMENTS

The author apologizes for so many self-citations, imposed by the very nature of the article. The author wishes to acknowledge all of the numerous contributions by colleagues (and friends) and from patients to what has been, retrospectively, a great journey.


Arthur Planul
email: arthur.planul@inserm.fr

Deniz Dalkara
email: deniz.dalkara@gmail.com

Vectors and Gene Delivery to the Retina

Keywords: adeno-associated virus, directed evolution, lentivirus, adenovirus, optogenetics, miRNA, promoter, nanoparticles

come

INTRODUCTION

There has been significant progress in discovering genes that are responsible for human retinal disease, and representative animal models carrying similar gene defects have been identified. In parallel, new methodologies like optogenetics that use light and genetics to manipulate and monitor the activities of defined neural populations have gained ground in asking questions on neuronal functioning and circuitry in an unprecedented way. With these advances, researchers started using retinal gene transfer extensively to develop gene-based therapies for blinding disorders and to investigate retinal circuitry. With the efficacy of gene delivery being central to the success of both gene therapy and understanding of retinal circuits, the development of adequate vectors has become critical. Therefore, there has been an exponential growth in the number of vectors for gene delivery to the mammalian retina in the past ten years. In this review, we discuss these vector technologies in view of their advantages and shortcomings for specific applications. More recently, several successful clinical gene therapy trials have paved the way to consider a number of retinal diseases with an expanded range of cellular targets for future therapies. In parallel, neuroscientists have moved on to understanding how different types of neurons contribute to circuit function in higher primates. There is now a growing demand to extend the vector toolbox for efficient gene delivery to retinas of higher primates. We discuss recent developments and research avenues in the development of vectors suitable for application in humans and higher primates in the Future Issues section.

GENE DELIVERY VECTORS

What Is a Vector?

In molecular biology, a vector is broadly described as a carrier that transfers genetic material into cells. This review focuses on vector systems for gene delivery to the mammalian retina. There are two main categories of vectors for gene delivery: viral and nonviral vectors. Viruses have evolved to efficiently deliver their genetic material into cells. Viral infection involves receptormediated intracellular uptake of viral particles and delivery of viral DNA into host cell nuclei, both of which are ensured by the viral capsid [START_REF] Kay | Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics[END_REF]. The viral capsids have coevolved with the constraints of their hosts to overcome and hijack the cell and nuclear entry mechanisms (Figure 1). For nonviral gene delivery, however, nuclear entry and persistent transgene expression are significant obstacles [START_REF] Naik | Gene delivery to the retina: focus on non-viral approaches[END_REF]). To improve nonviral gene delivery, chemical and molecular techniques need to be pushed toward a system that can approach the efficiency of natural biological processes of viral infection. In the unique environment of the retina, it might be possible to achieve this in the years to come. Nonviral vectors offer advantages over viral vectors in the size of the gene they can carry and in reduced risk of eliciting immune responses. Below, we discuss each type of vector in view of its potential for retinal gene delivery.

Synthetic Vectors

Synthetic vectors are often referred as nanoparticles (NPs) because of their small size (usually ranging from 10 to 500 nm). Synthetic vectors can be broadly defined as an assembly of cations that complex DNA into small-sized particles (Figure 2). NPs offer a unique set of advantages: They are easy to synthesize and usually have a low production cost compared to viruses (Trapani et al. 2014b). Their molecular structure can be easily manipulated. NPs also have a large transport capacity of up to 20 kb [START_REF] Fink | Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles[END_REF], which allows them to carry genes like ABCA4 or USH2A. Cellular entry of gene delivery vectors. The main steps in gene delivery are the cell entry, the escape from the endosome, and the entry into the nucleus for all vectors. The enveloped viruses reach the cytoplasm by membrane fusion, whereas the nonenveloped viruses and most of the synthetic vectors destabilize the endosome to escape into the cytoplasm. The DNA carried by synthetic vectors has difficulties entering the nucleus compared to viral DNA, except for CK30PEG, which is directly transported to the nucleus by nucleolin. DNA from lentivirus integrates randomly into the host genome. Rabies virus amplifies its RNA in the host cell, leading to the creation of new virions. Abbreviations: AAV, adeno-associated virus; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Transgene expression

Moreover, they possess a favorable safety profile due to their low immunogenicity and the absence of integration into the genome, alleviating insertional mutagenesis.

The key factors governing the efficacy of the NPs are (a) their cellular uptake (the NP should be able to protect the DNA in the extracellular medium and then to penetrate the cell), (b) their escape from the endosome into the cytosol, and (c) their ability to transfer the DNA through the nuclear pores, which block any particle bigger than ∼25 nm. There are different categories of synthetic vectors for gene delivery. This review focuses only on synthetic vectors that are able to transduce efficiently the neural retina or the retinal pigment epithelium (RPE) without toxicity [START_REF] Adijanto | Nanoparticle-based technologies for retinal gene therapy[END_REF].

CK30PEG is a 30-mer cationic polylysine conjugated with 10-kDa polyethylene glycol (PEG), which produces compacted DNA NPs. By using acetate as the lysine counterion, it is possible to obtain rod-shaped CK30PEG NPs efficient for retinal gene delivery [START_REF] Farjo | Efficient non-viral ocular gene transfer with compacted DNA nanoparticles[END_REF][START_REF] Fink | Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles[END_REF]. These vectors can efficiently transfect both the RPE and photoreceptor cells as early as 2 days postinjection into the subretinal space (Han et al. 2012b). Remarkably, their efficacy at 14 days postinjection (PI) is comparable to that of AAV2 and can last for up to 1 year. However, after 14 days PI, the expression levels resulting from the CK30PEG NPs' transduction become lower than for AAV2 and are strongly dependent on the promoter. CK30PEG NPs have the advantage of being internalized by nucleolin, which transfers the plasmid directly into the nucleus. Moreover, their small minor diameter (8-11 nm) seems to help their entry into the nucleus, infiltrating through the nuclear pores. CK30PEG NPs were capable of mediating structural and functional improvements in mouse models carrying mutations in photoreceptor-specific genes like the Abca4 -/-model of Stargardt disease (Han et al. 2012a). Polysaccharide NPs have also been used to deliver genes to the retina. Chitosan is a watersoluble product derived from chitin, the main structural component of crustacean exoskeleton and fungi cell wall. Chitosan NPs were shown to transduce photoreceptors and RPE after a subretinal injection and some retinal ganglion cells (RGCs) after intravitreal injection [START_REF] Puras | Oligochitosan polyplexes as carriers for retinal gene delivery[END_REF]. Nevertheless, the efficiency of these NPs seems relatively low. The chitosan NPs formed are cationic, but modifications of functional groups are often added to increase their solubility at neutral pH. Glycol-chitosan NPs have been shown to be able to transport the GFP gene under the control of two different promoters and transfect exclusively RPE cells after subretinal injections, without toxicity [START_REF] Mitra | Synthesis and characterization of glycol chitosan DNA nanoparticles for retinal gene delivery[END_REF]. This limitation to the RPE could be due to the formulation of the NPs, which formed a highly viscous gel that prevents the NPs from achieving a wide distribution into the retina.

Solid lipid nanoparticles (SLNs) are considered to be among the most effective lipid-based vectors. They consist of a solid lipid matrix surrounded by a layer of surfactants in an aqueous dispersion [START_REF] Battaglia | Application of lipid nanoparticles to ocular drug delivery application of lipid nanoparticles to ocular drug delivery[END_REF]). SLNs coupled with hyaluronic acid (HA-SLNs) transporting plasmids expressing GFP and RS1 were shown to transduce the RPE, the photoreceptors, and some RGCs. Remarkably, HA-SLNs transporting the human RS1 gene were able to improve the phenotype of a mouse model of X-linked juvenile retinoschisis [START_REF] Apaolaza | Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles[END_REF].

Despite progress in NP-mediated gene delivery, some challenges remain as there is high variability in transduction efficacy depending on the transported plasmid, and an increase in efficacy is often related to an increase in toxicity. All those problems lead to the conclusion that, for now, the synthetic vectors are not as efficient as viral vectors like adeno-associated viruses (AAVs) to deliver genes into the retina. However, the high transport capacities of the synthetic vectors leave more space for modification of the transported plasmid, thereby allowing the introduction of full promoter regions, introns, or scaffold/matrix attachment regions that can improve their efficacy [START_REF] Koirala | S/MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA[END_REF]).

Viral Vectors

The retina has several characteristics that make it a favorable tissue for gene delivery. First, accessing the retina is easy from a surgical standpoint. Second, injected vectors are trapped into the vitreous or under the retina, without any propagation into peripheral organs. This allows the retina to be immune privileged [START_REF] Willett | Immunology of AAV-mediated gene transfer in the eye[END_REF], thus diminishing risks of adverse effects due to an immune reaction against the viral vectors. Despite those advantages, the retina also has drawbacks limiting the use of certain vectors. The neurosensory retina is shielded by inner and external limiting membranes that restrict access of particles larger than 30 nm into the retina [START_REF] Inatani | Proteoglycans in retina[END_REF]. Thus, for gene delivery to the neural retina, AAV is the only vector that can be used via intraocular administration routes. AAVs are members of the Parvoviridae family of single-stranded DNA viruses. It is a dependovirus, which cannot replicate by itself, requiring coinfection with an adenovirus or a herpes simplex virus (HSV). AAV is a nonenveloped virus and has a large number of capsid variants displaying different cell tropisms [START_REF] Cearley | Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain[END_REF] (Figure 3). These are discussed in view of their utility for gene therapy or investigating retinal circuits in the next sections.

Vectors for probing the retina.

The visual system is a great model for probing the connectivity and function of neural circuits. With the mouse visual system being genetically traceable, it provides opportunities to understand these processes and link how identified circuit elements contribute to visual perception and behavior. Technologies like optogenetics are playing an important role in this process. Optogenetic actuators such as channelrhodopsin or halorhodopsin allow neuronal control, whereas a large spectrum of sensors like GCaMP synaptopHluorin, GluSnFRs, or ArcLightning allow optical recording of various aspects of neuronal activity. Viral and transgenic technologies enable restriction of both optogenetic control and optical recordings to genetically defined neuronal subpopulations.

The Gene Expression Nervous System Atlas (GENSAT) generated hundreds of mouse lines that express GFP or cre driven by different bacterial artificial chromosomes [START_REF] Heintz | BAC to the future: the use of bac transgenic mice for neuroscience research[END_REF]. Both GFP [START_REF] Tang | A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation[END_REF]) and cre [START_REF] Wang | Cre transgenic mouse lines[END_REF]) can be used in combination with viral vectors to confine expression of optogenetic actuators or sensors in the retina in a circuit-specific manner. A recombination event between two DNA recognition sites called loxP sites is catalyzed by crerecombinase. A DNA sequence in between loxP sites is said to be "floxed." The location and relative orientation of these loxP sites determine the product of cre-based recombination: DNA between oppositely oriented loxP sites will be inverted, whereas the sequence between loxP sites with the same orientation will be eliminated. For example, coinjection of AAVs transporting an inverted optogene sequence flanked by two loxP sites in a mouse line expressing cre in a subset of retinal neurons would allow specific delivery of the optogene into the retinal circuit. Furthermore, transgenic GFP reporter lines can be used for functional studies because of a method that transforms GFP into a molecule that enables genetic manipulation [START_REF] Tang | A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation[END_REF][START_REF] Tang | Cell type-specific manipulation with GFP-dependent Cre recombinase[END_REF]. In this system, a cre recombinase dependent on GFP (cre-DOG) is used to directly induce cre/loxP recombination in GFP-expressing cells. Using AAV viral vectors, cre-DOG can be delivered to multiple GFP mouse lines and induce effective recombination selectively in GFP-labeled cells. Nevertheless, the transgenic approaches using cre and GFP are mostly limited to mice. To go beyond the mouse model, viral vectors can be used in combination with cell type-specific promoters to target various subpopulations of retinal neurons. There are not enough characterized promoters to cover the diversity of retinal neurons as of today, but the discovery of new cell-specific promoters and enhancers [START_REF] Shen | Massively parallel cis-regulatory analysis in the mammalian central nervous system[END_REF] will open new avenues to expand our understanding of the neuronal circuitry of the visual system. Although AAVs remain the main viral vectors used within the retina, transsynaptic viruses like the rabies virus in combination with the retrograde HSV or AAVs are useful to probe the brain retina connections at a cellular resolution [START_REF] Yonehara | The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells[END_REF]. HSVs or AAVs can be used to transduce RGCs via their axon terminals in the lateral geniculate nucleus. This in turn allows the somas of the RGCs to be infected with a G-protein-deficient rabies virus coated with EnvA protein transporting a reporter protein (EnvA-G-GCaMP; Figure 4a). As the EnvA protein recognizes Pseudotyped rabies virus for studying the connections in the retina. (a) Normal and modified rabies virions.

Replacing the rabies G-protein gene with the GCaMP gene will lead to the production of GCaMPexpressing viruses and will allow the replacement of the G-protein at the surface of the virus by EnvA, which specifically recognizes the TVA receptor (EnvA-G-GCaMP). (b) Selective infection. Ganglion cells are transfected with two plasmids, one coding for the TVA receptor and one coding for the rabies G-protein.

Thus, only those cells expressing the TVA receptor will be recognized by EnvA and infected by the EnvA-G-GCaMP virions. (c) Monosynaptically restricted spread. The rabies G-protein coding plasmid will allow the newly produced rabies virions to be coated with G-protein, creating G-GCaMP virions able to cross one synapse in a retrograde fashion. As the synaptically connected cells do not have the G-protein coding plasmid, the new virions cannot cross one more synapse. The GCaMP will stain the TVA coated cells and the cells synaptically connected to the TVA coated cells (bipolar and amacrine cells). Adapted from [START_REF] Callaway | Transneuronal circuit tracing with neurotropic viruses[END_REF].

the TVA receptor exclusively, only the cells infected with the retrograde virus will express the activity reporter GCaMP (Figure 4b). Then, with the addition of the G-protein coding gene in those RGCs, the rabies virus is able to cross only one synapse and express GCaMP also in the presynaptic cells projecting to the RGCs [START_REF] Callaway | Transneuronal circuit tracing with neurotropic viruses[END_REF]; Figure 4c). This method has been used with success to study the origin of the direction selectivity (DS) in ON DS ganglion cells in mouse retinas [START_REF] Yonehara | The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells[END_REF]). However, it is difficult to use rabies viruses for long-term in vivo studies because of the toxicity of the virus, which modifies cell signaling and eventually leads to cell death (Granstedt el al. 2009).

Subretinal injection

Subretinal injection

Intravitreal injection

Intravitreal injection

RPE65, LRAT GNAT2 CNGB3 PRPH2 RS1 GUCY2D PDEb MYO7A RPGR CEP290 CRB1 Outer retina Inner retina AAV2 AAV2-7m8 ShH10 AAV8BP2 AAV2.3YF AAV2.4YF AAV2-7m8 ShH19 AAV2-7m8 AAV8BP2 AAV2.3YF AAV2.4YF AAV1-9 AAV2-7m8 AAV8BP2 AAV2.3YF AAV2.4YF

Photoreceptors

Retinal pigment epithelium

Horizontal cell

Bipolar cell

Müller glial cell

Amacrine cell

Retinal ganglion cells

Figure 5

Schematic showing the retinal cell types and injection routes. Cells in the inner and outer retina and the proteins encoded by candidate genes for gene therapy are indicated. Gene defects in the indicated genes lead to recessive phenotypes that are amenable to gene supplementation. Retinal pigment epithelium (dark red ), photoreceptors (red ), horizontal cell ( gray), bipolar cells (blue), M üller glial cell (orange), amacrine cell ( purple), retinal ganglion cells ( green).

Vectors for retinal gene therapy.

Blinding retinal diseases that can benefit from gene therapy include common multifactorial conditions like age-related macular degeneration, glaucoma, diabetic retinopathy, and inherited retinal degenerations due to single-gene defects. The complex diseases can benefit from gene therapy where a viral vector delivers a secretable factor alleviating the symptoms of the disease [START_REF] Maclachlan | Preclinical safety evaluation of AAV2-sFLT01-a gene therapy for age-related macular degeneration[END_REF]. In this case, cell type-specific gene delivery is not necessary. Monogenic retinal degenerations, however, are caused by mutations in any one of over 200 genes and can benefit from gene replacement therapy when the causal mutation is recessive. Recessive mutations causing inherited retinal degenerations (IRD) are found mostly in photoreceptor cells and RPE (Figure 5). Retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis, Stargardt disease, and achromatopsia are some of the most common and severe IRDs affecting photoreceptors [START_REF] Lipinski | Clinical applications of retinal gene therapy[END_REF]. Other monogenic conditions like congenital stationary night blindness and Leber hereditary optic neuropathy involve the inner retinal cells. The viral vectors used for gene therapy in these diseases need to have a favorable safety profile in addition to having coherent cell transduction patterns for the disease in question.

The choice of viral vectors for retinal gene therapy coalesces around adenoviral, lentiviral, and adeno-associated viral vectors. In general, when RPE is the target cell, all three vectors are capable of providing gene delivery (Trapani et al. 2014b), although lentiviral and adenoviral vectors are more inflammatory. Lentiviruses and adenoviruses can deliver genes exceeding the carrying capacity of AAVs. Nevertheless, the superior safety and efficacy of intraocular AAV administration in humans lead to efforts in engineering AAV-based vector systems for delivering large genes.

Dual AAV-mediated large gene delivery in mice (Trapani et al. 2014a) and in pigs [START_REF] Colella | Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors[END_REF] seems to be a viable solution to AAVs' limited cargo capacity.

TARGET CELLS IN THE RETINA

The majority of recessive mutations underlying inherited retinal degeneration are found in the outer retinal cells (RPE and photoreceptors) (Figure 5). The photoreceptors and RPE are thus the primary targets of gene replacement therapy. However, other recessive genetic mutations are also found in the M üller glial [START_REF] Pellissier | Gene therapy into photoreceptors and M üller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models[END_REF], bipolar [START_REF] Scalabrino | Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness[END_REF], and retinal ganglion cells [START_REF] Koilkonda | Leber's hereditary optic neuropathy-gene therapy: from benchtop to bedside[END_REF], making it necessary to find vectors that can target these cells successfully in a gene therapy setting. In addition to gene supplementation, other modes of gene therapy also require targeting inner retinal cells [START_REF] Busskamp | Optogenetic approaches to restoring visual function in retinitis pigmentosa[END_REF]. Finally, depending on the neuroscience question, it is equally important to be able to selectively and efficiently target all neuronal subtypes of the retina using viral vectors [START_REF] Packer | Targeting neurons and photons for optogenetics[END_REF]. The choice of AAV vectors that can be used to target the main retinal cell types are described below.

RPE Cells

The RPE cells have the particularity of being among the most actively phagocytic cells in nature [START_REF] Mazzoni | Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture[END_REF]. For this reason, they are easily targeted by virtually any viral vector administered into the subretinal space. AAV serotypes 1, 4, and 6 seem to have a preference for RPE cells rather than their underlying photoreceptors and should be used when exclusive RPE transduction is preferred [START_REF] Castle | Controlling AAV tropism in the nervous system with natural and engineered capsids[END_REF]. RPE cells can also be transduced via an engineered AAV variant, AAV2-7m8, when the vector is administered intravitreally at high doses [START_REF] Dalkara | In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous[END_REF].

Photoreceptors

Mature photoreceptors are efficiently targeted using AAV vectors administered into the subretinal space [START_REF] Allocca | Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors[END_REF][START_REF] Castle | Controlling AAV tropism in the nervous system with natural and engineered capsids[END_REF]. Several AAV serotypes and engineered variants have been shown to lead to high-level gene delivery to photoreceptors after subretinal injections. AAV serotypes 2, 5, 6, 7, 8, and 9 and engineered variants AAV2-7m8 and AAV8BP2 all allow efficient photoreceptor gene delivery [START_REF] Allocca | Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors[END_REF][START_REF] Boye | The human rhodopsin kinase promoter in an AAV5 vector confers rod-and cone-specific expression in the primate retina[END_REF][START_REF] Vandenberghe | Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey[END_REF][START_REF] Vandenberghe | AAV9 targets cone photoreceptors in the nonhuman primate retina[END_REF]. Among the naturally occurring serotypes, AAV8 leads to the highest overall photoreceptor transduction [START_REF] Castle | Controlling AAV tropism in the nervous system with natural and engineered capsids[END_REF]). AAV5, 8, and 9 have the advantage of giving rise to faster onset gene expression compared to AAV2 as well as gene expression in cone photoreceptors [START_REF] Alexander | Restoration of cone vision in a mouse model of achromatopsia[END_REF][START_REF] Komáromy | Gene therapy rescues cone function in congenital achromatopsia[END_REF][START_REF] Mancuso | Gene therapy for red-green colour blindness in adult primates[END_REF]. Photoreceptors can also be targeted via intravitreal injections of engineered AAV capsids [START_REF] Cronin | Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter[END_REF][START_REF] Dalkara | In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous[END_REF][START_REF] Kay | Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors[END_REF][START_REF] Petrs-Silva | High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors[END_REF], 2011). Directed evolution and rational design yielded AAV capsids capable of efficient outer retinal gene delivery from the vitreous.

Inner Nuclear Layer Neurons

The inner nuclear layer neurons (bipolar, horizontal, and amacrine cells) are the most difficult targets for gene delivery as they are separated from the vitreous and subretinal space by a layer of densely packed neurons. It has been impossible to target these neurons using naturally occurring serotypes. AAV engineering has provided solutions to reach these cells efficiently via subretinal [START_REF] Doroudchi | Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness[END_REF]) and intravitreal [START_REF] Gaub | Optogenetic vision restoration using rhodopsin for enhanced sensitivity[END_REF], Macé et al. 2014) routes.

M üller Glial Cell

One cell type that can be easily targeted within the inner nuclear layer is the M üller glial cell. M üller glia span the entire thickness of the retina with their end-feet localized in the inner and outer limiting membranes. Because of their unique morphology, they can be targeted with naturally occurring [START_REF] Allocca | Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors[END_REF][START_REF] Dorrell | Antioxidant or neurotrophic factor treatment preserves function in a mouse model of neovascularization-associated oxidative stress[END_REF]) and engineered [START_REF] Klimczak | A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat M üller cells[END_REF], Koerber et al. 2009) AAVs administered into the subretinal or intravitreal space. An AAV variant capable of targeting exclusive M üller glial targeting via the vitreous has been described and has the advantage of not requiring cell type-specific promoters to obtain restricted gene expression in this cell type [START_REF] Klimczak | A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat M üller cells[END_REF].

Retinal Ganglion Cells

Finally, the RGCs can be targeted with several AAVs via intravitreal injection route [START_REF] Caporale | LiGluR restores visual responses in rodent models of inherited blindness[END_REF], Dalkara et al. 2009[START_REF] Hellstr Öm | Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection[END_REF][START_REF] Ivanova | Evaluation of AAV-mediated expression of Chop2-GFP in the marmoset retina[END_REF][START_REF] Yin | Intravitreal injection of AAV2 transduces macaque inner retina[END_REF]. Contrary to subretinal injections, a limited number of AAVs lead to successful ganglion cell transduction despite the proximity of these cells to the injection site. One engineered AAV vector, AAV2-7m8, has been shown to these cells via subretinal injection [START_REF] Ramachandran | Evaluation of dose and safety of AAV7m8 and AAV8BP2 in the non-human primate retina[END_REF].

It is important to note that the tropism and capabilities of each AAV variant have been assessed by over a decade of experimental gene delivery studies, as overall capsid sequence homologies are not predictive of the tropism of AAV vectors. Only a few amino acid differences account for significantly different biological activities across the hundreds of naturally occurring and engineered AAV capsids [START_REF] Kwon | Designer gene delivery vectors: molecular engineering and evolution of adenoassociated viral vectors for enhanced gene transfer[END_REF].

INJECTION ROUTES

The administration route is a major determinant of efficacy and specificity in gene delivery to the retina. As a rule of thumb, the administration route that delivers the vector in closest proximity to the target cell type is preferred. This means that subretinal injections are relevant for gene delivery to the outer retina, whereas an intravitreal route is preferable for targeting the inner retina (Figure 5). However, the choice of delivery route can be more complex depending on the purpose of the gene delivery. Each gene delivery paradigm imposes a certain number of constraints. We focus on two main purposes for gene delivery: therapeutic gene delivery and gene delivery to investigate retinal circuitry.

For therapeutic gene delivery, it is necessary to choose the injection route that will afford therapeutic-level gene expression in target cells without causing toxicity or compromising retinal integrity. Indeed, it has been shown that disease state influences retinal transduction patterns using various viral vectors. In some diseases, retinal detachments are not well tolerated owing to the already compromised state of the retina. For investigating neural circuits, one can be more flexible regarding the safety, as long-term expression might not be necessary to adequately address a given question. The onset of gene expression is another parameter to consider when choosing an adequate delivery route. Subretinal injections of most serotypes lead to fast-onset gene expression in outer retinal cells starting at few days postinjection with the exception of AAV2, which has slower kinetics [START_REF] Tan | Gene therapy for retinitis pigmentosa and Leber congenital amaurosis caused by defects in AIPL1: effective rescue of mouse models of partial and complete Aipl1 deficiency using AAV2/2 and AAV2/8 vectors[END_REF]. Intravitreal injections lead to gene expression in the inner retina starting at 2-4 weeks postinjection depending on the injected dose. Transduction of the outer retina following intravitreal delivery is longer, varying from 6 to 8 weeks after administration [START_REF] Dalkara | In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous[END_REF]. Finally, gene delivery to the developing retina is possible via systemic injections of AAV (Byrne et al. 2015a[START_REF] Dalkara | Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9[END_REF]. When experimental conditions require gene expression before eyelid opening in mice (before day 15), delivering AAV through systemic injections into the bloodstream is less invasive and less challenging. High-titer injections of AAV9 and variants are able to provide bilateral gene delivery to inner and outer retina when the vector is administered within the postnatal period of 1 to 4 days (Byrne et al. 2015b[START_REF] Dalkara | Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9[END_REF]. Table 1 can be used to guide the choice of AAV for a given application.

BARRIERS TO RETINAL TRANSDUCTION USING VECTORS

Depending on the injection route, gene delivery vectors need to overcome certain obstacles to reach the retina. Vectors delivered into the subretinal space need to overcome the external limiting membrane of the photoreceptors to infiltrate the extracellular matrix surrounding the photoreceptors and gain access to the neurosensory retina. The major advantage of the subretinal injection route is that it concentrates the vector in close proximity to the retina. Conversely, intravitreal injections dilute the vectors in the vitreous. Vectors delivered into the vitreous cavity have to overcome the inner limiting membrane, which constitutes a formidable barrier to viral particles [START_REF] Dalkara | Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous[END_REF]. Inner limiting membrane is formed of a dense meshwork of glycosaminoglycans secreted by the M üller cell end-feet during development and isolates the retina from the vitreous. The ability of viral vectors to overcome this barrier depends on their size and the availability of their receptor within the inner limiting membrane. It has been shown that enzymatic lysis of this membrane of its physical peeling helps AAV penetrate better into the retina, increasing the efficacy of several serotypes in gaining access to the retina [START_REF] Cehajic-Kapetanovic | Glycosidic enzymes enhance retinal transduction following intravitreal delivery of AAV2[END_REF], Dalkara et al. 2009). The inner limiting membrane is much thicker in larger animals like dogs and macaques compared to rodents [START_REF] Matsumoto | Topographic variations in the rabbit and primate internal limiting membrane[END_REF]. Lastly, the blood-retina barrier limits the access of viral particles administered to the retina through the circulation. There seems to be an optimal time window when AAVs can get across the blood-retina barrier, and it is rather early in development (Byrne et al. 2015b).

The viral dose and the state of the retina are also important in determining the efficacy and extent of gene delivery to retinal cells exposed to the vector [START_REF] Kolstad | Changes in adeno-associated virusmediated gene delivery in retinal degeneration[END_REF][START_REF] Park | Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse[END_REF]). In general, retinal degeneration makes the retina more permissive to transduction by viral vectors as barriers limiting access to the retina are breached during the degenerative process. Viral dose acts as a gear on gene expression; the higher the dose, the more transgene expression there is in transduced cells. Some cells away from the injection site will actually be transduced with a limited number of viral particles, but expression is not visible in these cells because of the low copy number of the transgene in the nucleus. When viral dose is increased, this weak expression becomes visible, leading to an extended expression pattern.

RESTRICTING GENE EXPRESSION TO SUBPOPULATIONS OF NEURONS

The mammalian retina consists of millions of neurons, including dozens of neuronal subtypes. Retinal gene therapy aims to target these neurons in an efficient, safe, and specific way so that therapeutic gene expression can bring on benefit. Neuroscience studies, however, aim to understand the principles organizing retinal circuits and thereby decipher how they process information and account for vision. Recent developments suggest that genetic analysis will play an important role in dissecting neural circuits [START_REF] Shen | Massively parallel cis-regulatory analysis in the mammalian central nervous system[END_REF]. Informative analogies can be made between gene interaction networks that regulate complex biological processes and neural circuits. On the basis of this, the transgenes delivered into retinal cells must contain a promoter, which is a regulatory sequence that will determine where and when the transgene is active. Transgene expression after viral mediated gene delivery is a pentapartite equation between the injection route, the viral capsid, the state of the retina, the promoter, and the viral dose. The first four parameters are discussed above. The promoter is the transcriptional-level parameter determining which cells will express the transgene product. It is a critical sequence required for regulating the spatial and temporal expression pattern of a transgene delivered by a viral vector. Promoter sequences are isolated from upstream regions of endogenous mammalian genes. A promoter sequence normally includes a transcriptional start site as well as transcription regulatory sequences. Many promoters have been reported to successfully restrict expression of transgenes in the retina. Here, we focus on promoters that are compatible with delivery using AAV, as this is the only vector that can efficiently target the neurosensory retina. Table 2 provides a nonexhaustive list of promoter sequences that have been used with AAV vectors in the retina and their cell specificities.

However, one must remember that the specificity of a promoter is part of a larger equation involving the injection route, state of the retina, viral dose, and capsid. This means that ubiquitous promoters can also lead to cell type-specific expression when combined with a particular capsid and administration route, whereas cell type-specific promoters leading to restricted expression under a given set of circumstances may lose their specificity under others. Finally, the promoter activity also shows species-specific attributes.

CHALLENGES IN RETINAL GENE DELIVERY

Challenges for Basic Science Applications (in the Retina and Beyond the Retina Connectivity to Visual Centers)

There are three main challenges for the study of the retina with gene delivery vectors: the capacity to target the desired neuronal subtype, the ability to trace connectivity between the retina and visual centers without toxicity, and the transition to other, more visual species like nonhuman primates (NHPs).

We previously saw the importance of promoters specific to neuronal subtypes. As the AAVs are for now the only efficient viral vectors in the retina, the size of the promoter is limited because AAVs cannot carry more than 4.8 kb, including the promoter and the transgene. In the absence of promoter sequences that can distinctly target each neuronal subpopulation, the GENSAT project we described previously has been substantially useful. It brought an indirect way of obtaining restricted expression of genes in neuronal subtypes in the retina. It contributed to the study of the connectivity and activity of several neuronal subtypes of the retina like transient OFF-α and ON-OFF direction-selective RGCs [START_REF] Huberman | Architecture and activity-mediated refinement of axonal projections from a mosaic of genetically identified retinal ganglion cells[END_REF][START_REF] Huberman | Genetic identification of an On-Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion[END_REF]. However, the challenge remains the applicability of this approach in nonrodent species. It is of primary importance to be able to do circuit-specific interventions in NHPs to ask questions about our vision, which is remarkably different than the vision of rodents.

The second challenge is the study of the activity and the connectivity of the retina in relation to higher visual centers. For anterograde tracing of the visual system, the only available vectors are herpes simplex virus 1 (HSV1) [START_REF] Wojaczynski | The neuroinvasive profiles of H129 (herpes simplex virus type 1) recombinants with putative anterograde-only transneuronal spread properties HHS public access[END_REF] and AAV1 [START_REF] Zingg | AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors[END_REF]; however, HSV1 is toxic and will quickly modify the neuronal activity, and the genes transported by AAV1 need to be amplified by a system like cre/loxP to have a sufficient expression level. Moreover, these two vectors are not efficient in infecting the retina via intravitreal route. Thus, HSV1 and AAV1 can be used only via stereotaxic injection to study the visual area of the brain and not the connectivity within the retina. For retrograde tracing, rabies can be used. It is important to note that the immunogenicity of the vectors (i.e., rabies) is less of a problem in basic science applications, though toxicity could impair neuronal activity [START_REF] Granstedt | Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus[END_REF]. New viral vectors can help offer solutions in this context. For example, a new AAV called AAVretro has been developed to allow better retrograde transport in the brain [START_REF] Tervo | A designer AAV variant permits efficient retrograde access to projection neurons[END_REF]. This vector might also be relevant in studying the connectivity between the retina and the brain, although the length of the axons might pose a challenge. Because AAVs are much less toxic than typical transsynaptic viruses like rabies or HSV, they allow in vivo studies of neuronal activity in live animals. Once again, studies involving anterograde and retrograde tracing were carried out mostly on mice, and their applications to other species, particularly NHPs, remains problematic. Rabies [START_REF] Dum | Transneuronal tracing with neurotropic viruses reveals network macroarchitecture[END_REF] and lentivirus [START_REF] El-Shamayleh | Strategies for targeting primate neural circuits with viral vectors[END_REF] have been used in the brain and the spinal cord of macaques, but AAVs have been most frequently used for work in the retina [START_REF] Dalkara | In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous[END_REF][START_REF] El-Shamayleh | Strategies for targeting primate neural circuits with viral vectors[END_REF]. Finally, transgenic lines of marmoset are being developed [START_REF] Belmonte | Brains, genes, and primates[END_REF][START_REF] Sasaki | Generation of transgenic non-human primates with germline transmission[END_REF]), but their number remains largely inferior to rodents.

Challenges for Clinical Gene Therapy

A major challenge in bringing successful proof-of-concept gene therapies to the clinic is the lack of vectors tailored for gene delivery in human retinas. Preclinical gene therapy work has shown that viral vectors display substantially different transduction patterns in rodents and larger animals like dogs and NHPs [START_REF] Ivanova | Evaluation of AAV-mediated expression of Chop2-GFP in the marmoset retina[END_REF][START_REF] Lin | Characterization of engineered channelrhodopsin variants with improved properties and kinetics[END_REF][START_REF] Mussolino | AAV-mediated photoreceptor transduction of the pig cone-enriched retina[END_REF][START_REF] Ramachandran | Evaluation of dose and safety of AAV7m8 and AAV8BP2 in the non-human primate retina[END_REF][START_REF] Stieger | AAV-mediated gene therapy for retinal disorders in large animal models[END_REF][START_REF] Yin | Intravitreal injection of AAV2 transduces macaque inner retina[END_REF]. It is very likely that retinal transduction in humans will closely resemble those in NHPs. Much effort has been spent on making AAV vectors better in the context of mouse retina. It is now timely to invest AAV engineering efforts to generate vectors compatible with human use. Because transgene expression is dependent on not only the AAV capsid but also the promoter and disease state, efforts are also needed in adapting and testing promoters for their performance in NHP retinas. Lastly, generation of macaque models displaying hallmarks of retinal degeneration would be incredibly valuable for predicting the outcome of viral vector-mediated gene delivery in a clinical setting.

CONCLUSIONS

Both for gene therapy and for investigating the retina, huge progress has been made in the discovery and engineering of viral and synthetic vectors. The most adequate vector systems for efficient in vivo gene delivery today are viral vectors. AAV vectors are the only suitable vectors for gene delivery to the neurosensory retina and need to be coupled to cell type-specific promoters to give specific expression in a desired group of cells. Although transgenic lines are useful for obtaining cell type-specific expression in mice, new vector-promoter combinations are needed to target the primate retina. In particular, gene delivery vectors are not efficient in targeting the inner retina in primates because of the presence of a thick inner limiting membrane. A new set of tools needs to be developed to meet the needs of gene delivery in primates in the years to come.

  Figure 1

Figure 2

 2 Figure 2 Structural differences between synthetic vectors and viral vectors. (a) Rod-shaped CK30PEG nanoparticles with minor diameter of 8 to 11 nm. (b) The adeno-associated virus (arrowhead ) is smaller than the adenovirus (arrow), allowing it to cross the inner and outer limiting membrane after intraocular delivery. (c) The bullet-like rabies virus. Panel a adapted from Farjo et al. (2006). Panel b adapted from an image from the International Committee on Taxonomy of Viruses (http://www.virology.net/Big_Virology/BVDNAadeno. html). Panel c adapted from micrograph from F.A. Murphy, University of Texas Medical Branch, Galveston, TX (https://www.utmb. edu/virusimages/).

Figure 3

 3 Figure 3Organization of the adeno-associated virus (AAV) capsids. (a) Superposition of the VP1 protein of different AAV serotypes. AAV1 (red ), AAV2 (blue), AAV4 (cyan), AAV5 ( yellow), AAV6 ( green), AAV8 ( purple), AAV9 (magenta). (b) Hypervariable regions III, VIII, and IV. The structural differences are greatest among the nine hypervariable regions of the VP1 protein. This region determines the receptor-binding properties of the capsid and thus its tropism. (c) AAV2 capsid formed by 60 subunits assembled in an icosahedral fashion. The pentagon shows the place of the fivefold symmetry in the capsid. The triangle shows the place of the threefold symmetry; the more external regions of the capsid determine cellular tropism. These structures have been obtained using the UCSF Chimera software. The RCSB Protein Data Bank access numbers that have been used are PDB 5EGC (AAV1), PDB 1LP3 (AAV2), PDB 2G8G (AAV4), PDB 3NTT (AAV5), PDB 3OAH (AAV6), PDB 2QA0 (AAV8), and PDB 3UX1 (AAV9).

  Photoreceptor

Table 1 Retinal cell transduction with various capsids and injection routes Transduced cell types

 1

	Capsid	Intravitreal	Subretinal	Systemic
	AAV1	-	RPE, PR	-
	AAV2	RGC, MGC	RPE, PR	-
	AAV2.3YF	RGC, MGC, AC, HC	PR	-
	AAV2.4YF	RGC, MGC, AC, HC, PR, RPE	RPE, PR, HC, RGC	-
	AAV2-7m8	RGC, BPC, MGC, AC, HC, PR, RPE	RPE, PR, BPC, RGC	-
	AAV4	-	RPE	-
	AAV5	-	RPE, PR	-
	AAV6	RGC	RPE, PR	-
	AAV7	-	RPE, PR	-
	AAV8	RGC	RPE, PR	-
	AAV8BP2	RGC, BPC, AC, HC, PR	RPE, PR	-
	AAV9	RGC	RPE, PR, MGC	RPE, RGC
	AAV9.2YF	-	-	RPE, PR, MGC,
				RGC

Abbreviations: AC, amacrine cells; BPC, bipolar cells; HC, horizontal cells; MGC, M üller glial cells; PR, photoreceptors; RGC, retinal ganglion cells; RPE, retinal pigment epithelium.

Table 2 Targeting expression to subsets of retinal neurons

 2

	Promoter	Injection route	Animal model	Cell type	Reference(s)
	CMV	SR	Mouse, dog,	RPE, PR	Acland et al. 2001; Allocca et al. 2007;
			macaque		Auricchio et al. 2001, 2002; Lau et al.
					2000
		IVT	Mouse, dog,	RGC, MGC	Dudus et al. 1999, Guy et al. 1999,
			macaque, marmoset		Ivanova et al. 2010, Tshilenge et al.
					2016, Yin et al. 2011
	CBA	SR	Mouse	RPE, PR	Allocca et al. 2007
		IVT	Mouse, rat	RGC, MGC	McKinnon et al. 2002
	hSyn	IVT	Mouse	RGC	Caporale et al. 2011
	CD365	SR	Rat	RPE	Sutanto et al. 2005
	RPE65	SR	Mouse, dogs	RPE	Le Meur et al. 2007
	VMD2	SR	Mouse, rat, dog	RPE	Alexander & Hauswirth 2008, Conlon
					et al. 2013, Guziewicz et al. 2013
	MOPS	SR	Mouse	Rods	Flannery et al. 1997, Khani et al. 2007,
					Liu et al. 2005
	IRBPe/GNAT2	SR	Mouse, macaque	PR	Dyka et al. 2014
	hRK (GRK1)	SR	Mouse, pig	PR	Boye et al. 2010, Khani et al. 2007,
					Manfredi et al. 2013, Zou et al. 2011
	mBP	SR	Mouse	Cones	Michalakis et al. 2010
	HB	SR	Rat, dog	Cones (rods and	Glushakova et al. 2006, Komáromy et al.
				RPE)	2010
	RHO	SR	Mouse	Rods	Allocca et al. 2007
	mCAR	SR	Mouse	PR	Busskamp et al. 2010, Dyka et al. 2014
	pR2.1	SR	Mouse, rat, ferret,	Cones	Li et al. 2008
			guinea pig
	pR1.7	SR	Mouse, macaque	Cones	Ye et al. 2016
	GFAP	IVT	Mouse	MGC	Prentice et al. 2011
	Grm6	SR	Mouse	BPC	Doroudchi et al. 2011
	4xGrm6	SR, IVT	Mouse	BPC	Cronin et al. 2014, Gaub et al. 2015,
					Macé et al. 2015
	200En-	IVT	Mouse, marmoset	BPC	Lu et al. 2016
	mGluR500P
	In4s-In3-200En-	IVT	Mouse, marmoset	BPC	Lu et al. 2016
	mGluR500P
	Ple22	IVT	Mouse	BPC	de Leeuw et al. 2014, Scalabrino et al.
					2015
	Abbreviations: BPC, bipolar cells; IVT, intravitreal; MGC, M üller glial cells; PR, photoreceptors; RGC, retinal ganglion cells; RPE, retinal pigment
	epithelium; SR, subretinal.


Xuyang Chang
email: chang@lmt.ens-cachan.fr

Karine-Lavernhe Taillard

Olivier Hubert

UNI-AXIAL DISPLACEMENT CONTROLLED LOADING TEST OF SHAPE MEMORY NITI ALLOYS

Keywords: Displacement controlled loading #1, Shape memory alloy (SMA) #2, Digital images correlation

Introduction

Shape Memory Alloys (SMA) are a class of smart materials that undergoes an austenite-martensite solid-solid, isochoric, diffusion-less, 1st order phase transformation which confers its pseudo-elasticity (PE) and/or shape memory effect (SME) [START_REF] Lexcellent | Phase transformation yield surface determination for some shape memory alloy[END_REF] [START_REF] He | Rate-dependent domain spacing in a stretched strip[END_REF]. Phase transformation can be induced either by stress or temperature changes, thus indicating a strong thermomechanical coupled behavior. In industrial applications, SMA structures are on the other hand subjected to complex service conditions, such as large deformations, thermomechanical, multiaxial, and cyclic loading. Consequently, a robust multiphysic 3D modeling of phenomena governing theses behaviors is needed [START_REF] Depriester | Thermomechanical modelling of a NiTi SMA sample submitted to displacement-controlled tensile test[END_REF]. In order to construct a 3D thermal-mechanical coupled model, a campaign of 1D and 2D thermal-mechanical loading test is needed to identify the parameters. The loading test in uni-axial condition is especially addressed in this communication.

Displacement-controlled uniaxial tensile test experiments are performed over a small 1D NiTi strip to concentrate the stress level in the central zone and improve the heat exchange efficiency from the sample (due to phase transformation) to environment. Regularized Digital Images Correlation (R-DIC) technique is used to capture the temporal evolution of the displacement and strain fields [START_REF] Zvonimir | Mechanical-aided Digital Images Correlation[END_REF]. The influence of the presence of localization bands over the macroscopic stress-strain curve is firstly investigated. The local strain stress curve inside and outside the band are then compared to evaluate the homogeneity/heterogeneity of the strain field during the test and to conclude about the relevancy of this test.

Experimental Setups

1D strip

A textured poly-crystalline NiTi specimen is considered. The 1D strip specimen is a thin plate with a thickness e = 0.3 mm in the zone of interest with a rectangular shape : length =10 mm, width = 3 mm. This specific design permits to avoid local heat accumulation due to phase transformation during the loading process. In its zone of interest (ZOI), the sample's surface is covered by a black and white speckle pattern made of double internal mix airbrush (see Figure 1.a). The presence of the black and white pattern increases the gray level contrast inside the image. This condition is essential to conduct an accurate DIC analysis.

Loading set-up [5]

The machine used to apply the mechanical loading is shown below (Figure 1.b). This mini loading machine has 4 independent motors, that permit to apply any proportional or non-proportional displacement-controlled loading. Two cold-light sources generate a homogeneous lighting condition over the surface of the specimen. A camera is placed above perpendicular to the surface of the specimen to capture the image sequence at a given acquisition frequency.

1D test performed at 300K

Quasi-static displacement loading is applied to 1D NiTi strip at room temperature (T = 300K). Axial strain field is calculated by R-DIC analysis between reference image and deformed image. Axial tensile force is measured by the force cells of the machine.The average true axial strain and stress are calculated as follows: As we could see in Equation 2 and in Figure 2, the axial strain value is an average value of axial strains over all elements inside the chosen gauge of DIC. Average stress-strain behavior is plotted in Figure 3. Some remarkable points are highlighted and discussed below.

= 1 N i (1) σ = F s 0 (1 + ) (2

Evolution of the strain field

The strain field evolution is showed in Figure 4 and Figure 5 in eight steps. The eight steps correspond to points A to H reported Figure 3;

Point A : In the elastic regime, stress-axial strain behavior is linear and reversible, strain field remains small and its distribution is nearly homogeneous;

Local stress-strain curve inside and outside localization bands

In this subsection, we aim to compare the 'local' and macro stress and strain curve. In order to obtain different evaluations of the axial strain, three gauges (see Figure 6) are considered: Thus it means that gauge 1 will remain mostly inside the transformation zone from the beginning of test. On the contrary gauge 3 will stay nearly outside the localization bands for the most of the time during loading and unloading. This differentiation in terms of strain value, helps us focusing on the difference of the mechanical property inside and outside the band.

As we can see in Figure 6 and Figure 7, 'local' behaviors (in gauge 1 and 3) of the NiTi 1Dstrip differ from the macroscopic behavior (gauge 2). Gauge 1 has the smallest stress threshold for phase transformation, meanwhile zone gauge 3 exhibits the highest stress threshold. In order to better illustrate the spatial distribution of the strain field, the strain value along a specific line is plotted in Figure 8. The 4 different curves correspond to strain fields of Point A, B, C and D in Figure 3 and Figure 4.

As we can see , even inside the localization bands (strain field of Point D in Figure 3), there is a relative strong fluctuation in terms of the axial strain value (up to 10% variation). The heterogeneous feature of strain field inside the localization bands should be the main reason of the maximum axial strain value difference in Figure 6. Two different hypotheses can be proposed to explain the difference observed in stress transformation threshold :

Local diffused martensite phase formed during the elastic regime For gauge 1, during the elastic regime, a small martensite phase amount seems to be created during the 'elastic' regime. A relatively lower germination energy may consequently be required to bypass the transformation threshold (by a reduction of surface tension).

When the stress rises during the loading step, this zone is the first zone to fully transform from austenite to martensite. The localization bands begin to enlarge and propagate when the volume fraction of martensite is highly enough to equalize thermodynamic potential energy difference with other local zones. A combination of X-ray diffraction and Digital Images Correlation measurement may help to validate or invalidate this hypothesis. This work is in progress.

Structure effect As shown in Figure 8, the strain variation inside the localization bands remains relatively large. The difference in local and macroscopic behaviors could be the result of the combination of geometry effect (the smallest section is favored for nucleation -even considering a small geometrical defect) and heat conduction effect (temperature in zones aside the first nucleation area increases due to latent heat and conduction, even at small deformation rate). Implementation of the constitutive law in development inside a numerical modeling with implementation of geometry defect and heat equation resolution (including heat sources, conduction and convection) would help to validate or invalidate this hypothesis. This work is in progress.

Conclusions

The displacement-controlled uniaxial loading test over 1D strip of NiTi alloy helps us to understand the mechanical behavior of NiTi. DIC is a powerful tool which permits to capture the temporal evolution of the global strain field during the test, thus leading to a better understanding of the formation and propagation of the localization bands and hysteresis of NiTi alloy. By comparing the 'local' stress-strain curve for the zone inside and outside the localization, a difference of stress threshold in both loading and unloading is revealed.

Local diffused martensite during the elastic regime, temperature changes due to the phase transformation and multi-axiality of stress may cause these types of stress threshold difference.

To fully understand the reason of this stress threshold difference, two new tests need to be considered : Firstly, a stereo-hybrid measurement (synchronized infrared and optic camera acquisition) would help us to capture both the temperature and strain field at the same time, which helps to verify whether the temperature change is involved in this difference of the stress threshold. Secondly, a combination of X-ray diffraction and digital images correlation would permit to measure the volume fraction of each phase in this type of gauge. Indeed, through spatial scan of X-ray spot in the central zone, marteniste phase's volume fraction distribution field can be related to the strain field calculated by DIC. Until now, we are still working in both directions.

Figure 1 :

 1 Figure 1: 1.a 1D strip covered with a black and white speckle pattern 1.b Experimental setup

Figure 2 :

 2 Figure 2: Chosen gauge in the centre zone

Figure 3 :

 3 Figure 3: NiTi strip 1D stress strain curve at 300K

Figure 4 :Figure 5 :

 45 Figure 4: Evolution of the axial strain field during the loading

Gauge 1

 1 Local zone where the localization bands initiate; Gauge 2 Global central zone; Gauge 3 Local zone where the localization bands stop at the end of loading;

Figure 6 :Figure 7 :

 67 Figure 6: Three gauges of observation

Figure 8 :

 8 Figure 8: Spatial evolution of the local strain value along the chosen line