Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

Optimization of Capillary-Based Western Blotting for MYO7A.

Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5 kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.

Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6.

Cone-rod dystrophy 6 (CORD6) is caused by gain-of-function mutations in the GUCY2D gene, which encodes retinal guanylate cyclase-1 (RetGC1). There are currently no treatments available for this autosomal dominant disease, which is characterized by severe, early-onset visual impairment. The purpose of our study was to develop an adeno-associated virus (AAV)-CRISPR-Cas9-based approach referred to as "ablate and replace" and evaluate its therapeutic potential in mouse models of CORD6. This two-vector system delivers (1) CRISPR-Cas9 targeted to the early coding sequence of the wild-type and mutant GUCY2D alleles and (2) a CRISPR-Cas9-resistant cDNA copy of GUCY2D ("hardened" GUCY2D). Together, these vectors knock out ("ablate") expression of endogenous RetGC1 in photoreceptors and supplement ("replace") a healthy copy of exogenous GUCY2D. First, we confirmed that ablation of mutant R838S GUCY2D was therapeutic in a transgenic mouse model of CORD6. Next, we established a proof of concept for "ablate and replace" and optimized vector doses in Gucy2e+/-:Gucy2f-/- and Gucy2f-/- mice, respectively. Finally, we confirmed that the "ablate and replace" approach stably preserved retinal structure and function in a novel knockin mouse model of CORD6, the RetGC1 (hR838S, hWT) mouse. Taken together, our results support further development of the "ablate and replace" approach for treatment of CORD6.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.

ß-secretase 1 overexpression by AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration.

We reported previously that ß-site amyloid precursor protein cleaving enzyme (BACE1) is strongly expressed in the normal retina and that BACE1-/- mice develop pathological phenotypes associated with age-related macular degeneration (AMD). BACE1 expression is increased within the neural retina and retinal pigment epithelium (RPE) in AMD donor eyes suggesting that increased BACE1 is compensatory. We observed that AAV-mediated BACE1 overexpression in the RPE was maintained up to 6 months after AAV1-BACE1 administration. No significant changes in normal mouse visual function or retinal morphology were observed with low-dose vector while the high-dose vector demonstrated some early pathology which regressed with time. No increase in ß-amyloid was observed. BACE1 overexpression in the RPE of the superoxide dismutase 2 knockdown (SOD2 KD) mouse, which exhibits an AMD-like phenotype, prevented loss of retinal function and retinal pathology, and this was sustained out to 6 months. Furthermore, BACE1 overexpression was able to inhibit oxidative stress, microglial changes, and loss of RPE tight junction integrity (all features of AMD) in SOD2 KD mice. In conclusion, BACE1 plays a key role in retina/RPE homeostasis, and BACE1 overexpression offers a novel therapeutic target in the treatment of AMD.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis.

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.

Night vision restored in days after decades of congenital blindness.

Signaling of vision to the brain starts with the retinal phototransduction cascade which converts visible light from the environment into chemical changes. Vision impairment results when mutations inactivate proteins of the phototransduction cascade. A severe monogenically inherited blindness, Leber congenital amaurosis (LCA), is caused by mutations in the GUCY2D gene, leading to a molecular defect in the production of cyclic GMP, the second messenger of phototransduction. We studied two patients with GUCY2D-LCA who were undergoing gene augmentation therapy. Both patients had large deficits in rod photoreceptor-based night vision before intervention. Within days of therapy, rod vision in both patients changed dramatically; improvements in visual function and functional vision in these hyper-responding patients reached more than 3 log10 units (1000-fold), nearing healthy rod vision. Quick activation of the complex molecular pathways from retinal photoreceptor to visual cortex and behavior is thus possible in patients even after being disabled and dormant for decades.

Safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations.

A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.

Effects of Altering HSPG Binding and Capsid Hydrophilicity on Retinal Transduction by AAV.

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.ImportanceRationally 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.

Gene therapy reforms photoreceptor structure and restores vision in NPHP5-associated Leber congenital amaurosis.

The inherited childhood blindness caused by mutations in NPHP5, a form of Leber congenital amaurosis, results in abnormal development, dysfunction, and degeneration of photoreceptors. A naturally occurring NPHP5 mutation in dogs leads to a phenotype that very nearly duplicates the human retinopathy in terms of the photoreceptors involved, spatial distribution of degeneration, and the natural history of vision loss. We show that adeno-associated virus (AAV)-mediated NPHP5 gene augmentation of mutant canine retinas at the time of active degeneration and peak cell death stably restores photoreceptor structure, function, and vision with either the canine or human NPHP5 transgenes. Mutant cone photoreceptors, which failed to form outer segments during development, reform this structure after treatment. Degenerating rod photoreceptor outer segments are stabilized and develop normal structure. This process begins within 8 weeks after treatment and remains stable throughout the 6-month posttreatment period. In both photoreceptor cell classes mislocalization of rod and cone opsins is minimized or reversed. Retinal function and functional vision are restored. Efficacy of gene therapy in this large animal ciliopathy model of Leber congenital amaurosis provides a path for translation to human treatment.

Current Clinical Applications of In Vivo Gene Therapy with AAVs.

Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans. For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefit with a single treatment. This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity. There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials. The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic l-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders.

Site-specific modifications to AAV8 capsid yields enhanced brain transduction in the neonatal MPS IIIB mouse.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood-brain barrier. Modifications of adeno-associated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y444 + 733F) and a triple-mutant (Y444 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y-F + T-V) compared to AAV8 (double Y-F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y-F + T-V) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

SARM1 depletion rescues NMNAT1-dependent photoreceptor cell death and retinal degeneration.

Leber congenital amaurosis type nine is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking.

Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS.

BACKGROUND: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS: We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS: There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

A Novel Mouse Model of MYO7A USH1B Reveals Auditory and Visual System Haploinsufficiencies.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a - / - mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.