Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment

Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene replacement therapy for Leber’s congenital amaurosis type 2 establishes a platform for the development of downstream treatments targeting other forms of inherited and acquired ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene transfer technologies impede the development of gene replacement for each specific form of retinal degeneration. Here we describe gene augmentation strategies mediated by recombinant AAV vectors that impede retinal degeneration in pre-clinical models of acquired and inherited vision loss. We demonstrate distinct neuroprotective effects upon retinal ganglion cell survival and function in experimental optic neuritis following AAV-mediated gene augmentation. Gene transfer of the antioxidant transcription factor, NRF2, improves RGC survival while overexpression of the pro-survival and anti-inflammatory protein, SIRT1, promotes preservation of visual function. In the context of inherited retinal disease, we show stimulation of anabolic metabolism following AKT3 gene transfer preserves photoreceptor viability and delays functional loss in a mouse model of retinitis pigmentosa. In addition to these neuroprotective strategies, we also describe an approach to improve the in vitro potency of AAV vectors that are restricted by tissue-specific regulatory elements. This strategy utilizes genome engineering based on CRISPR/Cas9 gene activation to reprogram cell lines to specifically and potently express tissue-specific promoters of interest from AAV vectors

Development of a Novel Gene Therapy Using SIRT1 Signaling for Neuro-protection in Optic Neuropathies

Pharmacologic activators and genetic overexperession of sirtuin 1 (SIRT1) promote neuroprotective effects in multiple neurodegenerative disease models, including optic neuropathies. AAV2-SIRT1 mediated overexpression in mouse retina partially attenuates experimental optic neuritis despite only a 21% transduction efficiency for retinal ganglion cells (RGCs) using the AAV2-GFP vector for quantification. Viral vectors with increased transduction efficiency are needed

Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects. Keywords: gene therapy, molecular genetics, RNA editing, cell models, animal models, LCA10, CEP290, trans-splicin

Development of a Retinal Ganglion Cell Specific Gene Therapy Using SIRT1 Signaling for Neuro-Protection (Slides)

Loss of retinal ganglion cells (RGC) can occur by trauma, inflammatory, and ischemia leading to irreversible effects upon vision. Sirtuin 1 (SIRT1) deacetylase has demonstrated therapeutic value in multiple models of optic neuropathy with small molecule biologics. The initial use of gene therapy modestly improved visual outcomes in the EAE-induced mouse model of optic neuritis but with limited effects due to a low transduction rate and lack of cell specificity. Here we investigated the therapeutic potential of RGC specific SIRT1 gene augmentation in a mouse model of optic nerve crush (ONC)

Development of a Retinal Ganglion Cell Specific Gene Therapy Using SIRT1 Signaling for Neuro-Protection (PDF)

Loss of retinal ganglion cells (RGC) can occur by trauma, inflammatory, and ischemia leading to irreversible effects upon vision. Sirtuin 1 (SIRT1) deacetylase has demonstrated therapeutic value in multiple models of optic neuropathy with small molecule biologics. The initial use of gene therapy modestly improved visual outcomes in the EAE-induced mouse model of optic neuritis but with limited effects due to a low transduction rate and lack of cell specificity. Here we investigated the therapeutic potential of RGC specific SIRT1 gene augmentation in a mouse model of optic nerve crush (ONC)

Development of a Retinal Ganglion Cell Specific Gene Therapy Using SIRT1 Signaling for Neuro-Protection (Video)

Loss of retinal ganglion cells (RGC) can occur by trauma, inflammatory, and ischemia leading to irreversible effects upon vision. Sirtuin 1 (SIRT1) deacetylase has demonstrated therapeutic value in multiple models of optic neuropathy with small molecule biologics. The initial use of gene therapy modestly improved visual outcomes in the EAE-induced mouse model of optic neuritis but with limited effects due to a low transduction rate and lack of cell specificity. Here we investigated the therapeutic potential of RGC specific SIRT1 gene augmentation in a mouse model of optic nerve crush (ONC)