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The article focuses on adeno-associated virus (AAV)-mediated gene replacement which are been tested under clinical trials for treatment of inherited retinal disease including standards for using the AAV vector, introduction to AAV gene therapy and autosomal-recessive retinitis pigmentosa.

Background: Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model. Methods: Rd16;Nrl−/− mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA (n = 15; ages 7–48) were compared with those of the model. Results: CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe visual loss); the extent of this island declined slowly with age. The rd16;Nrl−/− model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant Nrl−/− mice, UV- and M-cone ERGs of rd16;Nrl−/− were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age. Conclusions: The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both CEP290-LCA patients and the rd16;Nrl−/− murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG. [ABSTRACT FROM AUTHOR]

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration. [ABSTRACT FROM AUTHOR]

Inherited retinal degeneration (IRD) disease and age-related macular degeneration (AMD) are leading causes of irreversible vision loss and blindness. Although significant progress has advanced the field in the past 5 years, significant challenges remain. The current article reviews the accomplishments and research advances that have fueled the development of treatments for patients with IRD and AMD, including the first approved gene-augmentation treatment for RPE65-related retinal degeneration and complement inhibition therapies to slow progression of geographic atrophy (GA) in AMD. The article outlines opportunities to address gaps and unmet needs that should lead to additional progress toward the development of treatments for patients with IRDs and non-neovascular AMD in the future.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1- GUCY2D in patients harboring biallelic recessive mutations in GUCY2D . Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1., Competing Interests: S.E.B. and S.L.B. are cofounders of Atsena Therapeutics, a company with related interests. They are recipients of research funding from Atsena Therapeutics., (© 2022 The Author(s).)

Blue cone monochromacy (BCM) is a congenital vision disorder affecting both middle-wavelength (M) and long-wavelength (L) cone photoreceptors of the human retina. BCM results from abolished expression of green and red light-sensitive visual pigments expressed in M- and L-cones, respectively. Previously, we showed that gene augmentation therapy to deliver either human L- or M-opsin rescues dorsal M-opsin dominant cone photoreceptors structurally and functionally in treated M-opsin knockout ( Opn1mw -/- ) mice. Although Opn1mw -/- mice represent a disease model for BCM patients with deletion mutations, at the cellular level, dorsal cones of Opn1mw -/- mice still express low levels of S-opsin, which are different from L- and M-cones of BCM patients carrying a congenital opsin deletion. To determine whether BCM cones lacking complete opsin expression from birth would benefit from AAV-mediated gene therapy, we evaluated the outcome of gene therapy, and determined the therapeutic window and longevity of rescue in a mouse model lacking both M- and S-opsin ( Opn1mw -/- /Opn1sw -/- ). Our data show that cones of Opn1mw -/- /Opn1sw -/- mice are viable at younger ages but undergo rapid degeneration. AAV-mediated expression of human L-opsin promoted cone outer segment regeneration and rescued cone-mediated function when mice were injected subretinally at 2 months of age or younger. Cone-mediated function and visually guided behavior were maintained for at least 8 months post-treatment. However, when mice were treated at 5 and 7 months of age, the chance and effectiveness of rescue was significantly reduced, although cones were still present in the retina. Crossing Opn1mw -/- /Opn1sw -/- mice with proteasomal activity reporter mice (Ub G76V -GFP) did not reveal GFP accumulation in Opn1mw -/- /Opn1sw -/- cones eliminating impaired degradation of ubiquitinated proteins as stress factor contributing to cone loss. Our results demonstrate that AAV-mediated gene augmentation therapy can rescue cone structure and function in a mouse model with a congenital opsin deletion, but also emphasize the importance that early intervention is crucial for successful therapy.

Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single amino acid (aa) mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding deficient capsid (AAV2ΔHS) lead to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction, and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model. Importance Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid (aa) mutations on retinal transduction as it relates to vector administration route. Through this approach we identified a largely deleterious relationship between hydrophilic aa mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic aa substitutions on a HSPG binding deficient capsid (AAV2ΔHS), generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases which require a large area of transduction., (Copyright © 2021 American Society for Microbiology.)

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative "subILM" injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.).

Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous group of diseases that account for the most severe form of early-onset retinal dystrophy. Mutations in retinal guanylate cyclase-1 (GUCY2D) are associated with LCA1, a prevalent form. GUCY2D encodes guanylate cyclase-1 (GC1), a protein expressed in rod and cone photoreceptors that regulates cGMP and Ca(2+) levels within these cells. LCA1 patients present with severely impaired vision, reduced, or ablated electroretinogram and nystagmus. Despite a high degree of visual disturbance, LCA1 patients retain normal photoreceptor laminar architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. This article will summarize clinical characterization of patients and proof of concept gene replacement studies in several animal models of GC1 deficiency, both of which have laid the groundwork for clinical application of a gene therapy for treatment of LCA1., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

Usher syndrome 1B (USH1B) is a severe, autosomal recessive, deaf-blind disorder caused by mutations in myosin 7A (MYO7A). Patients are born profoundly deaf and exhibit progressive loss of vision starting in their first decade. MYO7A is expressed in human photoreceptors and retinal pigment epithelium, but disease pathology begins in photoreceptors, highlighting the need to develop a gene replacement strategy that effectively targets this cell type. For its safety and efficacy in clinical trials and ability to transduce postmitotic photoreceptors, we have focused on developing a clinically applicable adeno-associated virus (AAV) platform for delivering full-length MYO7A cDNA (∼6.7 kb). Packaging of full-length MYO7A cDNA in AAV produces vectors with heterogeneous, fragmented genomes ("fAAV") capable of reconstituting full-length cDNA postinfection. We previously showed that fAAV vectors effectively delivered full-length MYO7A in vitro and in vivo. However, fAAV vectors are relatively inefficient and their heterogeneous genomes preclude definitive characterization, a drawback for clinical translatability. The aim of this study was to overcome these limitations by creating dual-AAV-vector platforms for USH1B with defined genomes. Human MYO7A was cloned in AAV vector pairs, each containing genomes <5 kb and intact inverted terminal repeats. One vector contained a promoter and 5' portion of the cDNA and the partner vector contained a 3' portion and polyadenylation signal. "Simple overlap" vectors share a central part of the MYO7A cDNA sequence. "Trans-splicing" and "hybrid" vectors utilize splice donor and acceptor sites with and without an additional central recombinogenic sequence, respectively. Vector pairs expressed full-length MYO7A in vitro and in vivo with equal or higher efficiency than fAAV, with a hybrid platform being most efficient. Importantly, analysis of MYO7A mRNA derived from each dual-vector platform revealed 100% fidelity to the predicted sequence. Our results suggest that dual AAV vectors with defined genetic payloads are a potential treatment option for USH1B.

Purpose: With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly for non-ocular tissues. A human retinal pigment epithelial cell line (ARPE19) and a mouse cone photoreceptor cell line, 661W, have been well characterized and are used for many retinal metabolism and biologic pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self-complementary (sc) adeno-associated virus (AAV) vectors in these biologically relevant ocular cell lines using high-throughput fluorescence-activated cell sorting (FACS) analysis., Methods: ARPE19 and 661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at multiplicity of infections (MOIs) ranging from 100 to 10,000. Three days post infection fluorescent images verified mCherry expression. Following microscopy, FACS analysis was performed to quantify the number of positive cells and the mean intensity of mCherry fluorescence, the product of which is reported as transduction efficiency for each vector. The scAAV vectors containing cone-specific (sc-mCARpro-green fluorescent protein [GFP]), rod-specific (sc-MOPS500-eGFP), retinal pigment epithelium (RPE)-specific (sc-VMD2-GFP), or ubiquitous (sc-smCBA-GFP) promoters were used to infect both cell lines at an MOI of 10,000. Three days post infection, cells were immunostained with an antibody raised against GFP and imaged. Finally, based on our in vitro results, we tested a prediction of transduction efficiency in vivo., Results: Expression from unmodified scAAV1, scAAV2, scAAV5, and scAAV8 vectors was detectable by FACS in both ARPE19 and 661W cells, with scAAV1 and scAAV2 being the most efficient in both cell lines. scAAV5 showed moderate efficiency in both ARPE19 and 661W cells. scAAV8 was moderately efficient in 661W cells and was by comparison less so in ARPE19 cells; however, transduction was still apparent. scAAV9 performed poorly in both cell types. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with the increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases, efficiency improved; in others, it was unchanged or marginally reduced. Retinal-specific promoters were also active in both cell lines, with the 661W cells showing a pattern consistent with the in vivo activity of the respective promoters tested. The prediction based on in vitro data that AAV2 sextuple Y-F mutants would show higher transduction efficiency in RPE relative to AAV2 triple Y-F capsid mutants was validated by evaluating the transduction characteristics of the two mutant vectors in mouse retina., Conclusions: Our results suggest that this rapid and quantifiable cell-based assay using two biologically relevant ocular cell lines will prove useful in screening and optimizing AAV vectors for application in retina-targeted gene therapies.

Purpose: Dramatic restoration of retinal function has followed subretinal viral-mediated gene therapy in RPE65-deficient animal models of human Leber congenital amaurosis (LCA) caused by RPE65 mutations. Progress in early-phase clinical trials of RPE65-LCA prompted us to begin development of an in vivo bioassay of clinical grade vector stability for later-phase trials., Methods: Naturally-occurring Rpe65-mutant rd12 mice (2-4 mo of age) were studied with full-field electroretinograms (ERGs). Flash stimuli (range, -4.1 to 3.6 log scot-cd x s x m(-2)) were used to evoke ERGs in anesthetized, dark-adapted mice. B-wave amplitudes were measured conventionally and luminance-response functions were fit. Leading edges of photoresponses were analyzed with a model of rod phototransduction activation. A unilateral subretinal injection of AAV2-CB(SB)-hRPE65 vector was delivered and therapeutic efficacy of 4 doses spanning a 2 log unit range was studied with ERGs performed about 6 weeks after injection. Uninjected rd12 eyes and wild-type (wt) mice served as controls., Results: Rd12 mice showed substantially smaller amplitudes and lower sensitivities than wt mice for all measured ERG b-wave and photoresponse parameters. For the dose-response study, there was no difference between 0.01X-dosed mice and untreated mutants. Improved receptoral and post-receptoral function was evident for 0.1X, 0.3X, 1X doses: b-wave semi-saturation constants decreased, b-wave amplitudes increased with dose; photoresponses showed faster kinetics and higher maximum amplitudes. ERG b-wave amplitude to a selected stimulus light intensity could provide evidence of biologic activity of the vector; interocular differences in b-wave amplitude comparing treated versus untreated eyes in the same animal also revealed vector efficacy., Conclusions: We have taken the first steps toward developing an ERG assay of biologic activity of human grade vector for future clinical trials of RPE65-LCA. Faithful murine models of treatable human disease tested with specific ERG protocols may emerge as valuable in vivo bioassays for future human clinical trials of therapy in many retinal degenerative diseases.