Novel Adeno-Associated Viral Vectors for Retinal Gene Therapy

REVIEW Novel adeno-associated viral vectors for retinal gene therapy

This article has been corrected since Advance Online Publication and an erratum is also printed in this issue

LH Vandenberghe1 and A Auricchio2,3

Vectors derived from adeno-associated virus (AAV) are currently the most promising vehicles for therapeutic gene delivery to the retina. Recently, subretinal administration of AAV2 has been demonstrated to be safe and effective in patients with a rare form of inherited childhood blindness, suggesting that AAV-mediated retinal gene therapy may be successfully extended to other blinding conditions. This is further supported by the great versatility of AAV as a vector platform as there are a large number of AAV variants and many of these have unique transduction characteristics useful for targeting different cell types in the retina including glia, epithelium and many types of neurons. Naturally occurring, rationally designed or in vitro evolved AAV vectors are currently being utilized to transduce several different cell types in the retina and to treat a variety of animal models of retinal disease. The continuous and creative development of AAV vectors provides opportunities to overcome existing challenges in retinal gene therapy such as efﬁcient transfer of genes exceeding AAV’s cargo capacity, or the targeting of speciﬁc cells within the retina or transduction of photoreceptors following routinely used intravitreal injections. Such developments should ultimately advance the treatment of a wide range of blinding retinal conditions. Gene Therapy (2012) 19, 162–168; doi:10.1038/gt.2011.151; published online 13 October 2011

Keywords: AAV; adeno-associated virus; retina; subretinal; intravitreal; eye gene therapy

INTRODUCTION virus, and potentially block vector transduction.8 These concerns Adeno-associated virus (AAV) was first identified as a potentially prompted a search for alternatives for AAV2 that could overcome useful vector for gene transfer in the early 1980s by pioneering work the deficits, yet preserve the benefits of the platform. This was on AAV serotype 2 in the laboratories of Drs Carter and Muzyczka.1–4 facilitated by the genetics of AAV in that it allowed cross-packaging Several aspects of the natural biology of the virus appeared attractive of AAV2-based genomes with an alternative capsid shell.9 for somatic gene transfer; the virus is not associated with human disease, its small genome was molecularly cloned and could fairly AAV CAPSID SEROTYPES, ISOLATES AND MUTANTS: NATURE readily be manipulated in vitro, and the virus is able to transduce VERSUS NURTURE terminally differentiated, non-dividing cells.5 The exploration of AAV AAV contains a single-stranded DNA genome packed in an icosahedral as a gene transfer vector highlighted even more interesting properties; capsid comprised of three structural Cap proteins (VP1, -2 and -3). The AAV requires only the terminal repeats in cis for genome packaging particle is largely made up by numerous copies of the 60 kDa AAV and particle assembly, eliminating all viral-coding DNA from the VP3 protein. However, for every 10 VP3 molecules, one VP1 and one vector genome and thereby reducing its immunogenicity and freeing VP2 capsid monomer (whose C-terminal sequence is identical to up more room for genetic cargo. And, as was established in later VP3) is anchored in the virion architecture.5 The first alternative research, every base pair counts, as AAV’s size limitation is one of its capsids that were evaluated by cross-packaging with AAV2 genomes biggest limitations for its use in gene therapy, including its ocular were the capsids from various natural serotypes, many of which were applications. isolated sometime between 1950 and 1990 and molecularly cloned The potential benefits of AAV vectors justified their early applica- over the last 10 years (reviewed in Gao et al.7). These serotypes, AAV1 tion in clinical trials of gene therapy for cystic fibrosis and hemophilia through six (sometimes referred to as the AAV 6-pack), provided B.6 However, these trials did not demonstrate functionally significant remarkable potential compared with AAV2 in terms of tropism and levels of gene transfer, and there was no evidence for therapeutic or transduction efficiency in murine models, for example, AAV1 in the clinical benefit. AAV serotype 2, though an excellent gene transfer muscle and AAV5 in the central nervous system. Motivated by these vector for transducing certain cells (for example, hepatocytes, the findings, the group of Dr James M Wilson at the University of retinal pigment epithelium (RPE) or retinal ganglion cells), was found Pennsylvania, isolated latent AAV from non-human primate (NHP) to be too inefficient for transducing many other cell types, for and human tissues by PCR and identified over 120 novel AAV example, photoreceptors (PRs).7 Second, AAV2 neutralizing antibo- isolates clustered in six viral clades, four of which were previously dies, directed to epitopes on the viral capsid, were found to be uncharacterized. Three serotype vectors, AAV7, 8 and 9 and some of prevalent in human population due to prior exposure to the natural the vectors based on isolates from these studies provided even greater

1Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA; 2Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy and 3Medical Genetics, Department of Pediatrics, Federico II University, Naples, Italy Correspondence: Dr LH Vandenberghe, Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA. E-mail: [email protected] or Professor A Auricchio, Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy. E-mail: [email protected] Received 31 August 2011; accepted 1 September 2011; published online 13 October 2011 LH Vandenberghe and A Auricchio 163 potential in terms of levels of transgene expression in several important et al.21), but only a few groups have brought this approach to the therapeutic targets such as the liver, muscle and retina. Other groups retina, or cell types relevant to the retina. The groups of David Schaffer have focused attention on non-primate sources of AAV and have and John Flannery did, however, take this approach to identify AAVs characterized AAVs endemic in cow, pig, bird, goat and mouse.10–14 with improved glial tropism from a panel of libraries generated in his A growing number of AAV serotypes have been resolved structu- laboratory. Multiple rounds of selection on cultured primary human rally15,16 and the improved understanding of the structure-function astrocytes yielded a number of candidate vectors, which next relationship of the virion has provided the means to develop rational underwent confirmatory screening in vivo for their ability to target approaches to altering the capsid in silico and in vitro. A primary astrocytes in the rat central nervous system. These experiments yielded ambition of these strategies is to tailor the vector for a particular two AAV2 variants named ShH19 and L1-12 and the AAV6-like therapeutic goal, for example by enhancing its gene transfer efficiency, ShH10 with substantially more astrocyte transduction in comparison altering its cell specificity, improving its safety profile or combining to AAV2 and AAV6 controls. These observation led them to desired phenotypes from more than one serotype. This ambition is explore the potential of these vectors in the retina where ShH10 shared by an another method of capsid engineering, referred to as demonstrated an improved tropism for Mu¨ller cells via intravitreal directed evolution of AAV. Briefly, in directed evolution, a large library injection.22,23 of laboratory-generated capsid variants is subjected to pressure by applying conditions selective for the desired vector phenotype. The AAV TARGETING IN THE RETINA: A FUNCTION OF DOSE, technique thereby does not require a thorough understanding of ADMINISTRATION ROUTE, DISEASE STATE, ANIMAL MODEL, AAV’s structure-function biology or protein engineering, as it is AND CAPSID unbiased in its approach. Both directed evolution and rational design The viral capsid is not the only determinant of the specificity or strategies for AAV were reviewed elsewhere,8 and here we will restrict efficacy of retinal cell transduction; several other factors affect our discussion to those engineered constructs that have been evaluated AAV-mediated gene transfer to the retina. AAV vectors often have a in the retina. broad tropism, but their targeting can be steered by their route of First, in a rational design effort, the Srivastava group aimed at administration or site of delivery. This is true for the retina where the intracellular redirection of AAV particles away from the proteasome, two main vector delivery approaches are intravitreal and subretinal to accomplish higher levels of transduction at lower doses. Basic injection. The intravitreal route deposits the vector dose in the trafficking studies suggested that capsid tyrosine residues could be vitreous.24 With the advent of anti-angiogenic therapies that are phosphorylated, pre-destining them for ubiquitination and eventual administered through intravitreal injections, these procedures are proteosome degradation. On the basis of the available structural data now very routine clinical ophthalmologic interventions. Intravitreal on AAV serotypes, site-directed mutagenesis of one or more tyrosines injection of AAV2 vectors results in efficient transduction of retinal to phenylalanine indeed increased gene transfer efficiency in various in ganglion cells.25 No other AAV serotypes (at least those with unmo- vivo and in vitro settings.17,18 Initial proof-of-concept of this exciting dified capsids—see later) appear to transduce the retina following approach was established with AAV2, but has now been extended to intravitreal injection. A second type of vector delivery deposits the multiple other serotypes including AAV8.18 Data from these studies vector below the retina, in a virtual space between the RPE and the PR demonstrated in murine models marked enhancement of retinal called the subretinal space. Subretinal administrations of vectors transduction following intraocular delivery. Which tyrosine residue induce a regional retinal detachment called bleb, which flattens within mutation is optimal for an individual serotype requires empirical hours or days following vector uptake and diffusion of the diluent.24 evaluation but for AAV2-Y444F, AAV2-Y730F, AAV8-Y733F and In humans and large animals the procedure is usually performed with AAV9-Y446F a reduced dose requirement, broader tropism within a (partial) vitrectomy to provide more volume for the vector-contain- the neuronal retina and enhanced diffusion across the retina were ing solution and to relieve ocular pressure. In rodents, it is not noted in the mouse model.18 More recently, AAV2 variants with possible to carry out a vitrectomy, but subretinal injections are multiple tyrosine mutations further demonstrated improvements in performed adequately without them. Subretinal injections, both retinal transduction and diffusion following subretinal and intravitreal clinically and in large and small animal models, are technically more delivery.19 Importantly, an AAV8-Y733F encoding guanosine mono- challenging than intraviteal injections and have a higher potential for phosphate-phosphodiesterase was shown to rescue long-term function surgical morbidity. However, to date it is the most efficient method for and structure rescue of disease in rd10 mice, a particularly challenging targeting PRs and RPE cells, which are the most relevant cell types for model for autosomal recessive RP.20 A second example of the potential treating many forms of inherited blindness. Subretinal injection of any of structure-function based engineering of the AAV capsid came from of the AAV serotypes tested to date can result in very efficient the study of the wealth of sequence information obtained on the AAV transduction of RPE (usually 100% transduction in the area of virome. Unique capsid residues were identified in AAV isolates from detachment) and variable transduction of PRs (between 20 and 80% human and NHP tissues that were not shared by homologous AAVs. depending, on the serotype and titre used26—see later). Although the These proviral singleton residues were thought to be failed attempts by growing experience in vitreo-retinal surgery raised the acceptability of the virus to enhance its fitness and this hypothesis was challenged by subretinal injections as a procedure for gene therapy applications an altering them to the residue shared by its related partners.8 In some intravitreal approach, which distributes vector, allow more homoge- cases, singleton-reversed particles demonstrated improved transducing nous transduction and pose a lower risk of surgical complications, ability, but its main effect was seen in marked increases in titer, would be greatly preferred. For this reason the field is actively likely due to increased structural stability during viral assembly. pursuing vectors that enable transduction across the retina from the Taken together, these rational design approaches expanded the AAV vitreal side. Advances are being made in the understanding of the toolbox with a set of dozens of novel gene transfer reagents that require structural and biochemical barriers that must be overcome to make to evaluation in all relevant therapeutic target organs, including the retina. make this happen. Dalkara et al.23 demonstrated elegantly how the Many groups have pursued directed evolution as a means to select vitreoretinal junction is a serotype-specific hurdle for AAV, and AAVs derived from combinatorial libraries (reviewed by Perabo the authors speculate the abundance of AAV serotype receptors on

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the inner limiting is a factor in limiting the diffusion across this with transduction of the contralateral eye in mice (at higher doses), barrier.18 Novel vectors, such as the multiple tyrosine mutants of this was not observed in dogs or NHPs. AAV2,19 are emerging for which diffusion does not appear to be AAV vector transduction characteristics may also be impacted by hampered and they show significant potential to transduce RPE and the host species and the health of the retina. This is exemplified by two PR following intravitreal injection in small animals. However, it remains sets of observations. First, cone PR targeting by AAV2 has been to be seen whether the efficiencies of gene transfer are sufficient for described in the canine retina.40 However, in NHPs cone transduction therapeutic gene delivery to these cells, and if intravitreal injection of was not observed even at higher doses, indicating a species-specific these vectors is effective in large animal models where there are more serotype tropism.41 Second, broad transduction of the neuronal retina pronounced physical barriers between the vitreous and inner retina. In via an intravitreal route of injection is difficult and remains a major addition, recent studies highlight an increased risk of systemic vector challenge for the field, although intravitreal injection of AAV2 and dissemination and immune activation following intravitreal adminis- AAV8 vectors in a murine model of retinoschisis have been shown to tration of vector, possibly limiting its use for gene therapy.27,28 Of the result in robust transduction of the neuroretina, probably due to several clinical trials, underway or completed, that are using AAV2 to disrupted retinal lamination. The contrasting results in these examples treat retinal diseases, three have used subretinal injection to target are likely attributable to anatomical and/or biochemical differences RPE cells in order to treat a form of Lebers congenital amaurosis between hosts that affect vector diffusion and or the availability of the (LCA2)27,29,30 and one is using intravitreal injection for the delivery of cognate receptor for the particular AAV serotype. In an elegant report, an anti-angiogenic molecule, sFLT, aimed at treating the neovascular Flannery’s laboratory described how differences in the structural and complications of age-related macular degeneration.28 biochemical makeup between normal and diseased retinas impact the With several translational programs underway in AAV-based ocular diffusion of AAV serotypes following intravitreal injection. Data shows gene therapy, increased emphasis is being placed on determining dose that the cell types in inner retina become much more accessible for requirements, therapeutic thresholds and dose-limiting toxicity. In AAV during retinal degeneration,23 an observation that they attribute human trials, the maximum reported dose that has been used to date to the concomitant disorganization of the inner limiting membrane is 1.5Â1011 particles and was found to be safe and well tolerated and (ILM).23 However, this does not apply to all types of retinal in the absence of notable cellular or humoral immune responses to degeneration as outer retina targeting via intravitreal administration vector or transgene.31 In animals, much higher doses have been of AAV is poor in the rd10 model of retinitis pigmentosa.42 administered, in part to achieve the highest level of transgene expression and in part to target less permissive retinal cell types. From a GETTING TO THE RETINAL SWITCH: AAV-MEDIATED PR literature survey, the highest dose injected subretinally in animal TRANSDUCTION models is 2.7Â1013 particles for NHP32 and 6.4Â1013 for dogs,33 Inherited retinal degenerations (IRDs) with an overall prevalence of both, though not specifically monitored for, in the apparent absence 1/2000 worldwide43 are a major cause of blindness and affect over of immune sequelae. Other studies, in both NHP and dogs, indicate 200 000 people in the European Union. Among the most frequent and that elevated AAV doses do lead to toxic or inflammatory responses to severe forms of IRD are recessive retinitis pigmentosa (RP), Leber the gene transfer, although it is unclear whether this is related to vector congenital amaurosis (LCA) and Stargardt disease (SD). RP, LCA and dose or the high level of expression.26,34 In mice and rats subretinal SD, as with other forms IRDs, are characterized by loss of neuronal injections of doses as high as 5Â1010 and 1Â1012 particles,35 respec- PR, rods and/or cones in the retina.44 Independent of the disease tively are well tolerated. A subretinal dosing study with AAV2 and causing mutations, PR loss occurs by apoptosis.44 The majority of AAV8 in NHPs highlighted the importance of dose and vector mutations causing IRDs occur in genes expressed in PR.44 Thus targeting; low doses targeted RPE exclusively where higher doses efficient gene transfer to PR is key for treatment of most of these were required to transduce PRs. Cone PRs prove to be particularly blinding conditions. The first evidence that AAV targets PR in refractory to AAV transduction, which can be overcome in part by addition to the RPE was provided by the group of Robin Ali.45 elevating vector dose26 and switching serotype. Lastly, an important Since then, several groups have shown that AAV2 transduces PR of dose-related aspect of translating retinal gene therapies to the clinic, is various species including NHP26,41 and dogs46 upon subretinal repeat administration. Most forms of retinal degeneration, particularly administration. Indeed, therapeutic efficacy, albeit transient, has the inherited forms, affect both eyes and ideally, will require a bilateral been observed upon AAV2 subretinal administration in rds mice, a injection. There are a number of parameters that make this procedure model of RP47 and in other IRD models48–50 due to mutations in quite complex. First, two injections doubles the dose of vector genes expressed in PR. The possibility of swapping capsids with ease administered to the subject. Second, unless the injections are between different AAV serotypes provided access to a portfolio of performed simultaneously, which is impractical and unlikely, the novel recombinant AAV vectors, and the search for improved PR first injection, and the nature of the immune response thereof, may targeting AAVs began. In mice, AAV5 immediately appeared superior impact the safety and/efficacy of the second. Finally, the interval to AAV2(refs 9, 52, 53) as it fairly efficiently targeted PR at higher levels between the first and the repeat injection is anticipated to have a than was previously seen with AAV2. Also, its onset of expression was role qualitatively as well as quantitatively in terms of host response to faster by about a week. The group of Beverly Davidson has shown that the second injection. This complexity poses a difficulty for both AAV5 transduces efficiently NHP rod, but not cone PR.53 Soon, robust clinician–investigators and regulators to navigate through as is evi- proof-of-concept was provided of AAV5-mediated gene supplementa- denced by a recent symposium organized by the US Food and Drug tion therapy in animal models of IRD caused by defects in genes Administration Advisory Committee on Cellular, Tissue and Gene expressed in PRs.54,55 The Hauswirth group has additionally demon- Therapies on this topic.36 Ocular re-adminstration of AAV has been strated the ability of AAV5 to transduce cone PR with elevated doses studied for bilateral subretinal injection in mouse,37 dog38 and and cone-specific promoters in dogs33 and NHP32 models of achro- monkey.39 Overall, these studies show safe and efficient gene transfer matopsia. Despite some promising short-term improvements,56 gene of both eyes for the conditions tested. Neutralizing antibodies to AAV supplementation using AAV5 has not been very effective at rescuing do arise following the first injection, but where these may interfere the severe PR degeneration found in the rd10 murine model of RP, in

Gene Therapy LH Vandenberghe and A Auricchio 165 which PR degeneration is due to partial deficiency of the beta subunit products produced by over sized AAV5 preparations mainly result of phosphodiesterase, a key enzyme in the phototransduction from the re-assembly of truncated packaged genomes66 or are derived cascade.57 The contrasting results achieved with AAV5 in the different from a minor subset of vectors in which large genomes have been disease models likely illustrate AAV5’s ability to target PR but does so observed (Sommella and Auricchio, unpublished results) has yet to be insufficiently efficient for the more severe and progressive models of demonstrated. Irrespective of the mechanism by which the large retinal degeneration such as in the rd10 mice. This may be due to the transgene products are generated, the high heterogeneity of oversized difficulty in achieving sufficient or appropriate levels of transgene AAV genomes remains a major limitation, frustrating efforts to expression despite efficient levels of PR transduction. In recent years, progress this promising approach towards the clinic. AAV8 has emerged, among the naturally-occurring AAV serotypes, as providing the highest levels of PR transduction in several species. In AAV-MEDIATED GENE TRANSFER TO THE RPE: THE FIRST mice, we and others demonstrated AAV8’s superior ability to trans- CLINICAL SUCCESS OF OCULAR GENE THERAPY duce PRs efficiently, fast and delivering high transgene levels as Virtually all AAV serotypes tested by subretinal administration compared with AAV5.58,59 We also demonstrated that AAV7, and to transduce the RPE efficiently. Whether this is due to an inherent a lesser extent AAV9, are good candidates for targeting PRs in the permissiveness of the RPE to AAV infection (that is, the presence of mouse.59 A side-by-side comparison in pigs60 demonstrated higher PR AAV receptors and co-receptors at the RPE cell membrane) or is due transduction with subretinally administered AAV8 versus AAV5. High to the phagocytic properties of the RPE that could facilitate entry of levels of AAV8-mediated PR transduction have been further observed AAV particles is not clear. However, efficient AAV2-mediated RPE in dogs.61 In NHP, these findings from mouse, pig and dog were transduction in the Royal College of Surgeon rat, a model of RP basically confirmed and highlighted AAV8’s potential for PR gene caused by deficient RPE phagocytic activity, partly argues against this targeting; AAV2 also demonstrated PR targeting but did so only at a latter hypothesis.67 Although the majority of AAV serotypes transduce 10-fold higher dose than AAV8. At moderate doses, rods, but not additional cell types in addition to the RPE (that is, AAV2, 5, 7 and 8 cones, are efficiently transduced by AAV8; whereas at higher doses rod transduce PR,9,45,59 AAV1 transduces cells in the INL9), AAV2/4 and to some extent also cones are targeted.26 The level of PR transduces exclusively the RPE of various species.68 preservation in rd10 mice following AAV8-mediated gene supplemen- Several proof-of-concept studies of AAV-mediated correction of tation therapy was superior to that obtained using an AAV5 vector,42 RPE-specific IRDs have been provided in the last decade (for review although the rescue was still relatively modest compared with those see Stieger et al 69). Among these, the most successful have been the achieved using AAV5 vectors in more stationary disorders.54,55 More restoration of vision obtained following subretinal administrations of effective rescue of the rd10 mouse was achieved in a study using AAV8 AAV2 in the Briard dog model of LCA type 2 (LCA2) due to vectors with Tyr to Phe mutagenesis of some capsid residues. mutations in RPE65, encoding the RPE-specific all-trans retinol Although this study achieved the first long-term morphological and isomerase.46 This extraordinary result initially obtained by a group functional rescue of the rd10 mouse,62 it was not clear whether this of investigators coordinated by Jean Bennett at the University of was due to improved efficacy due to the altered capsid, or whether it Pennsylvania and then replicated by several other groups70–72 has was because the treated animals were dark reared, which slows opened the doors to three independent clinical trials testing the safety degeneration substantially; there was no direct comparison using and efficacy of subretinal administrations of AAV2 in LCA2 AAV8 vectors with unaltered capsids. Other studies using AAV8 patients.27,29,30 Although a detailed discussion of the results from vectors include very effective gene supplementation therapy in a these trials is beyond the scope of this review, some important mouse model of achromatopsia due to CNGB3 deficiency63 and a conclusions can be drawn from these studies: (i) AAV2 subretinal mouse model of LCA type 1, caused by GC1 deficiency.64 AAV8 administration appears safe and well tolerated; (ii) AAV2 subretinal vectors have also been used for gene supplementation therapy to administrations resulted in improvement of visual function in all trials rescue function and slow degeneration in a mouse model of LCA type although at different levels; (iii) in one trial there was evidence that the 4causedbytheAipl1deficiency.50 The Aipl1À/À mouse has the most maximal efficacy was obtained in the youngest LCA2 patients, severe PR degeneration of any model, and rescue of this model using presumably with better retinal preservation.31 Overall these results an AAV8 vector perhaps provides the strongest indication that AAV8 bode well for the development of AAV-based therapies for additional vectors might be superior to other vectors for transduction of RPs. RPE-specific defects. Although yet to be established, clinical proof-of-concept rescue of PR-specific IRD appears achievable given the levels of PR transduction TRANSDUCTION BEYOND THE ILM: THE INNER RETINA AND AAV and rescue obtained in animal models to date. Already early on, in some of first in vivo AAV gene transfer experi- A major remaining challenge is using AAV, a vector well known for ments to the retina, it was observed that following intravitreal its limited cargo capacity, to mediate the safe and efficient delivery of injection AAV2 had the ability to transduce the retinal ganglion cells large genes (that is, 45 kb in size) to PRs. Attempts have been made (RGC) lining the vitreous.25 And that is where the field remained for to package into AAV capsids cDNAs from genes mutated in IRDs several years; indeed, the first serotype comparison studies indicated exceeding the wild type AAV genome size, that is, ABCA4 (mutated in that AAV2 but not AAV1 or AAV5 could target the retina from the SD) and MYO7A (mutated in Usher Syndrome type IB).65 Subretinal vitreal side.9,73 In addition to RGCs, AAV2 also quite extensively administrations of these ‘over sized’ AAV vectors derived from AAV5 transduced the trabecular meshwork and occasionally Mu¨ller cells.9 In resulted in expression of proteins of the expected size and in subsequent studies that included more of the natural serotypes and significant phenotypic improvement of a mouse model of SD, the isolates, AAV6 emerged as another vector with potential to transduce most common form of inherited macular degeneration in humans. the retina from the vitreal side.74 Interestingly, these serotypes both However, the mechanism underlying over sized AAV-mediated PR bind heparin.75,76 Contrary to AAV2, in a quantitative study in the rat, transduction remains elusive as the genomes contained in over sized AAV6 appears to distribute deeper in the inner nuclear layer where it AAV vectors appear highly heterogeneous in size and are predomi- can transduce amacrine, bipolar and Mu¨ller cells including those nantly shorter than expected.66 Whether ABCA4 or MYO7A transgene lining the retinal blood vessels.74,77 Mu¨ller glia are present throughout

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the entire retinal thickness and are vital to the neurons they are differences in transduction properties (or vice versa). In order to embedded in; they nourish, recycle neurotransmitters and photo- facilitate the use of AAV as a clinically-versatile vector for the pigments, maintain osmotic homeostatis and protect against oxidative treatment of IRD, irrespective of the causative gene or the severity stress. Mu¨ller cells have a role in many forms of retinal degeneration of retinal atrophy, it is essential to identify the appropriate AAV and become activated under stress, which eventually may lead to serotypes for targeting each of the major retinal cell types in humans. gliosis.78 Their central role in the retina makes them an important To this end, in addition to in vivo administration to NHP, human target for gene therapy. Other vectors with reported affinity for the retinal organ culture is attracting increasing interest as a tool to study inner retina are AAV isolates rh.8 and rh.10.79 As mentioned pre- AAV vector tropism and transduction characteristics.82 Ultimately, viously, data is emerging that the ILM is a structural and biochemical one can envisage a scenario in which extensive efficacy testing in small barrier for AAV in a serotype-dependent manner, possibly due to the animal models will no longer be required: one may develop a differential sequestration of AAV serotypes on the ILM by the presence therapeutic AAV vector based on the most efficient serotype selected of their cognate receptors.23 These studies indicate a much broader in NHP or human retinal culture, test its potency (that is, ability to pattern of expression with the natural AAV serotypes when the ILM is express the therapeutic transgene) in explants and directly progress to perturbed during the progression of retinal degeneration23 or by an production of vector under Good Manufacturing Practice standards enzymatic digestion with Pronase E.23 The authors argue that the and assessment of safety and toxicity before going into humans. This is barrier to transduction is unlikely to be diffusional or purely physical clearly a discussion for scientists and regulators to address urgently, because some viruses, including some AAV serotypes, are able to cross particularly for those IRDs that are lacking, or for which there are the ILM fairly efficiently. Surgical or other routes to remove, bypass or sub-optimal, animal models. avoid the ILM may indeed be the key for achieving efficient transduc- Although most of the focus in recent years has been placed on tion via an intravitreal route. transduction of RPE and PR cells, an additional desirable target would The observation that some AAVs can, and some cannot penetrate be cells in the inner retina, such as bipolar neurons. Interest in inner the inner retina, validates approaches to alter the vector and thereby retinal cells has increased because the recent observation that avoid the blocking of transduction by the ILM. Directed evolution for AAV-mediated gene transfer of microbial rhodopsins to inner retinal glial tropism, yielded several promising new vector candidates for cells83,84 or to light-insensitive cones,85 which are viable long after rods Mu¨ller glia targeting in the retina.22,80 The rational design strategy of are lost in conditions such as retinitis pigmentosa, induce light- tyrosine modifications to the AAV capsid to improve transduction activated responses and restore vision-driven behavior. This exciting properties of existing serotypes has proven promising in this respect, approach, best described as a molecular prosthetic, in which one with significant enhancement of AAV2’s ability to target RGCs and reprograms a cell to become light sensitive, may provide a gene-based Mu¨ller cells. In addition, in mouse, a few amacrine and rod bipolar therapeutic alternative for patient populations with complete loss of cells can be targeted with these improved vectors.18 Tyrosine PR that would not benefit from more classical gene augmentation modifications to AAV8 and AAV9 vectors also result in improved approaches. The ability of the small and penetrating AAV vectors to transduction following intravitreal injection, although AAV2 variants target the outer retina from the vitreal side may be an additional still appear to remain superior via this route. Single residue changes in characteristic to further advance both pre-clinical and clinical retinal these vectors, enabled transduction in a murine model at levels gene therapy given the ease of intravitreal administrations and the exceeding those of AAV2.18 At higher doses, these vectors also diffuse pattern of transduction that a single intravitreal administration demonstrate potential to penetrate to the outer retina and even the can achieve. Finally, the efficient transfer of large genes to the retina RPE to deliver their genetic payload.19 It remains to be seen, however, using AAV vectors remains a challenge especially given that strategies whether these findings translate to larger animal models in which the such as those based on over-sized AAV vectors may not be clinically ILM is considerably thicker compared with rodents. accepted until the high heterogeneity in genome size is reduced. Each of these challenges may be overcome by the development of CONCLUSIONS AND PROSPECTS AAV vectors with the appropriate capsid, the key determinant for The last decade has seen exciting advances in the field of AAV vector AAV-mediated transduction. Rational design, directed evolution or in development and application. The identification of dozens of natu- vivo phage bio-panning86,87 in which affinity peptides are inserted in rally-occurring AAVs81 and the parallel development of AAV variants the AAV capsid have proven to be powerful methods for vector based on either directed evolution21 or rational mutagenesis17 have engineering. Ultimately, cell-restricted gene expression will be created efficient gene transfer tools for a variety of target cells and required, preferably with cell-specific targeting by AAV, rather than tissues including the retina. Thanks to these developments, even the with the currently used methods of transcriptional or post-transcrip- most aggressive types of retinal degeneration may become treatable. tional88 control of gene expression. The demonstration of safety and efficacy of intraocular AAV admin- In conclusion, the future of AAV-mediated retinal gene transfer istration in humans has provided a unique momentum for retinal appears bright as the increasing arsenal of AAV variants, whether gene therapy to move into the clinical arena. Although these results natural or artificial, is proving an invaluable source of gene transfer fuel the field with enthusiasm, some challenges remain, which will vehicles for each specific need. require additional technological developments to be overcome. Rigorous comparison of the ability of the various AAV serotypes CONFLICT OF INTEREST and variants to transduce specific cell types should be carried out in LHV is co-inventor of AAV technologies including novel serotypes large animal models predictive of the behavior in the human retina which are described in patents licensed to various pharmaceutical and (ideally the NHP as it is the only model with a macula). As expensive biotechnological companies. as this appears, it is a crucial step for the translation of the proof-of- concept work established in the often-used smaller animal models of ACKNOWLEDGEMENTS retinal diseases. Certain AAV serotypes, whereas efficient in these We thank Dr Therese Cronin and Dr Robin Ali for the discussion and critical models, could actually be clinically irrelevant due to inter-species reading of the manuscript. Supported in part by: Grant Number UL1RR024134

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