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Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.Graphic abstract

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa. Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening of 68,979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection of 25 pmol MTX increased electroretinogram response and rhodopsin level in the retinae of Rho(P23H/+) mice at one month of age. Additionally, four weekly intravitreal injections increased the photoreceptor cell number in the retinae of Rho(P23H/+) mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin associated retinitis pigmentosa.

AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.

The delivery of AAV vectors across physical barriers to target the outer retina is key to the success of retinal gene therapies. Subretinal injection of AAV vectors is the most common approach to deliver genes to photoreceptors and RPE; however, this route of administration presents surgical challenges and potential complications, as well as mediates transfer to only a limited region of the retinal surface. Intravitreal administration may represent a safer way of targeting larger areas of the outer retina; physical barriers such as the internal limiting membrane significantly limit efficiency of natural AAVs through this route. In a prior murine proof of concept study, we showed that directed evolution is promising method for engineering new AAV capsid variants with enhanced permissivity for the outer retina. Here, we show results from a screen performed in the canine retina, a large animal model with eye structure and size similar to the human eye. A library of >107 distinct capsid variant AAV vectors was injected intravitreally into the eye of a wildtype dog. A subset of the AAV vectors injected was recovered from samples of outer retina by PCR amplification. Isolated variants were then pooled and re-injected in additional wildtype canine eyes, and following 5 total rounds of selection the pool had converged to a relatively small number of variants. High throughput sequencing was used to characterize the convergence of variants over all rounds of selection. The final candidate variants were then evaluated by packaging a GFP reporter controlled by a ubiquitous CAG promoter. Retinal distribution and cellular tropism of the variants was examined by cSLO (AF mode) imaging, histology and immunocytochemistry. Nineteen variants were identified as candidate vectors for targeting the canine outer retina. Intravitreal injection of a mixture of equal amounts of these variants carrying GFP fused to a barcode resulted in efficient targeting of all retinal layers including photoreceptors and RPE in 6 eyes of 3 wildtype dogs. Thus, directed evolution performed in a large animal model, with an eye structure similar to that of humans, resulted in new AAV capsid variants with improved abilities to bypass significant structural barriers and efficiently transduce the outer retina. High throughput sequencing allowed for identification of promising variants and monitoring of convergence. These vectors show promise for gene therapy delivery via the intravitreal route of administration, potentially providing a safer approach for targeting a larger area of outer retina than currently achieved by the more traumatic and focal subretinal injection method.

© 2015 The American Society of Gene & Cell Therapy. Systemic delivery of AAV9 offers the potential for widespread and efficient gene delivery to the retina, and may thus be a useful approach for treatment of disease where intraocular injections are not possible, for syndromes affecting multiple organs, or where early intervention is required. The expression resulting from intravenous injection of AAV9 is more efficient in neonates than adults, and here we characterize the effect of age on retinal transduction of AAV9 in the mouse retina. We find that the pattern of expression in neonatal mice is correlated to the development of the retinal vasculature, and that the area of the retinal transduction as well as the cell types infected vary depending on the age at injection. Furthermore, we demonstrate that sequential injections of AAV9 vectors carrying two different transgenes infect adjacent areas of the retina, providing a larger area of coverage. Lastly, we show that the retina's endogenous spatiotemporal expression pattern of Mfsd2a, a protein associated with the maturation of a functional blood-brain barrier, coincides with suppression of retinal transduction by intravenously-delivered AAV9, suggesting that AAV9 crosses the blood-retina barrier through transcytosis.

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.