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Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken ß-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

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SIRT1 prevents retinal ganglion cell (RGC) loss in several acute and subacute optic neuropathy models following pharmacologic activation or genetic overexpression. We hypothesized that adeno-associated virus (AAV)-mediated overexpression of SIRT1 in RGCs in a chronic ocular hypertension model can reduce RGC loss, thereby preserving visual function by sustained therapeutic effect. A control vector AAV-eGFP and therapeutic vector AAV-SIRT1 were constructed and optimized for transduction efficiency. A magnetic microbead mouse model of ocular hypertension was optimized to induce a time-dependent and chronic loss of visual function and RGC degeneration. Mice received intravitreal injection of control or therapeutic AAV in which a codon-optimized human SIRT1 expression is driven by a RGC selective promoter. Intraocular pressure (IOP) was measured, and visual function was examined by optokinetic response (OKR) weekly for 49 days following microbead injection. Visual function, RGC survival, and axon numbers were compared among control and therapeutic AAV-treated animals. AAV-eGFP and AAV-SIRT1 showed transduction efficiency of ~ 40%. AAV-SIRT1 maintains the transduction of SIRT1 over time and is selectively expressed in RGCs. Intravitreal injections of AAV-SIRT1 in a glaucoma model preserved visual function, increased RGC survival, and reduced axonal degeneration compared with the control construct. Over-expression of SIRT1 through AAV-mediated gene transduction indicates a RGC-selective component of neuroprotection in multiple models of acute optic nerve degeneration. Results here show a neuroprotective effect of RGC-selective gene therapy in a chronic glaucoma model characterized by sustained elevation of IOP and subsequent RGC loss. Results suggest that this strategy may be an effective therapeutic approach for treating glaucoma, and warrants evaluation for the treatment of other chronic neurodegenerative diseases.

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Optic neuritis (ON), the most common ocular manifestation of multiple sclerosis, is an autoimmune inflammatory demyelinating disease also characterized by degeneration of retinal ganglion cells (RGCs) and their axons, which commonly leads to visual impairment despite attempted treatments. Although ON disease etiology is not known, changes in the redox system and exacerbated optic nerve inflammation play a major role in the pathogenesis of the disease. Silent information regulator 1 (sirtuin-1/SIRT1) is a ubiquitously expressed NAD+-dependent deacetylase, which functions to reduce/prevent both oxidative stress and inflammation in various tissues. Non-specific upregulation of SIRT1 by pharmacologic and genetic approaches attenuates RGC loss in experimental ON. Herein, we hypothesized that targeted expression of SIRT1 selectively in RGCs using an adeno-associated virus (AAV) vector as a delivery vehicle is an effective approach to reducing neurodegeneration and preserving vision in ON. We tested this hypothesis through intravitreal injection of AAV7m8.SNCG.SIRT1, an AAV2-derived vector optimized for highly efficient SIRT1 transgene transfer and protein expression into RGCs in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis that recapitulates optic neuritis RGC loss and axon demyelination. Our data show that EAE mice injected with a control vehicle exhibit progressive alteration of visual function reflected by decreasing optokinetic response (OKR) scores, whereas comparatively, AAV7m8.SNCG.SIRT1-injected EAE mice maintain higher OKR scores, suggesting that SIRT1 reduces the visual deficit imparted by EAE. Consistent with this, RGC survival determined by immunolabeling is increased and axon demyelination is decreased in the AAV7m8.SNCG.SIRT1 RGC-injected group of EAE mice compared to the mouse EAE counterpart injected with a vehicle or with control vector AAV7m8.SNCG.eGFP. However, immune cell infiltration of the optic nerve is not significantly different among all EAE groups of mice injected with either vehicle or AAV7m8.SNCG.SIRT1. We conclude that despite minimally affecting the inflammatory response in the optic nerve, AAV7m8-mediated SIRT1 transfer into RGCs has a neuroprotective potential against RGC loss, axon demyelination and vison deficits associated with EAE. Together, these data suggest that SIRT1 exerts direct effects on RGC survival and function.

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SIRT1 prevents retinal ganglion cell (RGC) loss in models of optic neuropathy following pharmacologic activation or genetic overexpression. The exact mechanism of loss is not known, prior evidence suggests this is through oxidative stress to either neighboring cells or RGC specifically. We investigated the neuroprotective potential of RGC-selective SIRT1 gene therapy in the optic nerve crush (ONC) model. We hypothesized that AAV-mediated overexpression of SIRT1 in RGCs reduces RGC loss, thereby preserving visual function. Cohorts of C57Bl/6J mice received intravitreal injection of experimental or control AAVs using either a ganglion cell promoter or a constitutive promoter and ONC was performed. Visual function was examined by optokinetic response (OKR) for 7 days following ONC. Retina and optic nerves were harvested to investigate RGC survival by immunolabeling. The AAV7m8-SNCG.SIRT1 vector showed 44% transduction efficiency for RGCs compared with 25% (P > 0.05) by AAV2-CAG.SIRT1, and AAV7m8-SNCG.SIRT1 drives expression selectively in RGCs in vivo. Animals modeling ONC demonstrated reduced visual acuity compared to controls. Intravitreal delivery of AAV7m8-SNCG.SIRT1 mediated significant preservation of the OKR and RGC survival compared to AAV7m8-SNCG.eGFP controls, an effect not seen with the AAV2 vector. RGC-selective expression of SIRT1 offers a targeted therapy for an animal model with significant ganglion cell loss. Over-expression of SIRT1 through AAV-mediated gene transduction suggests a RGC selective component of neuro-protection using the ONC model. This study expands our understanding of SIRT1 mediated neuroprotection in the context of compressive or traumatic optic neuropathy, making it a strong therapeutic candidate for testing in all optic neuropathies.

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Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-ß (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.

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Patient-derived human-induced pluripotent stem cells (iPSCs) have been critical in advancing our understanding of the underlying mechanisms of numerous retinal disorders. Many of these retinal disorders have no effective treatment and result in severe visual impairment and even blindness. Among the retinal degenerative diseases modeled by iPSCs are age-related macular degeneration (AMD), glaucoma, Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and autosomal dominant retinitis pigmentosa (adRP). In addition to studying retinal disease ontogenesis and pathology, hiPSCs have clinical and pharmacological applications, such as developing drug screening and gene therapy approaches and new cell-based clinical treatments. Recent studies have primarily focused on three retinal cell fates - retinal pigmented epithelium cells (RPE), retinal ganglion cells (RGCs), and photoreceptor cells - and have demonstrated that hiPSCs have great potential for increasing our knowledge of and developing treatments for retinal degenerative disorders.

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Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements.

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Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

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Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM mutant cells. We then identified a recombinant adeno-associated virus (AAV) serotype, AAV7m8, that is optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed AAV7m8.hCHM, which delivers the human CHM cDNA under control of CMV-enhanced chicken ß-actin promoter (CßA). Delivery of AAV7m8.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia.

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Human retinal degeneration can cause blindness, and the lack of relevant model systems has made identifying underlying mechanisms challenging. Parfitt et al. (2016) generate three-dimensional retinal tissue from patient-derived induced pluripotent stem cells to identify how CEP290 mutations cause retinal degeneration, and show an antisense approach can correct disease-associated phenotypes.

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