Ocular Gene Therapy

National Eye Institute

Investigators

Abstract

1. We have continued to provide fast, responsive and high quality AAV vector construction and packaging services to NEI intramural researchers as well as investigators in other NIH institutes. Below is a list of projects performed in the current fiscal year. AAV produced: Number Date AAV plamid madeby plasmid cloned by. Made for PI 1 8/19/22 AAV2 CMV hNRL_AS321_s50T OGTC OGTC NNRL Anand Swaroop 2 8/19/22 AAV8 RK tdTomato_U6 Rd9gRNA OGTC OGTC NNRL Anand Swaroop 3 8/25/22 AAV2 CMV hNRL_AS321_P51s OGTC OGTC NNRL Anand Swaroop 4 8/25/22 AAV2 CMV EGFP OGTC OGTC NNRL Anand Swaroop 5 12/15/22 AAV5 hVMD2 Serpine3 OGTC OGTC Robert Hufnagel 6 12/15/22 AAV2 CMV hNRL_AS321_G122E OGTC OGTC NNRL Anand Swaroop 7 12/15/22 AAV2 CMV hNRL_AS321_H125Q OGTC OGTC NNRL Anand Swaroop 8 7/6/23 AAV8 CMV hNRL_AS321 OGTC OGTC NNRL Anand Swaroop 9 7/6/23 AAV2 CMV EGFP OGTC OGTC NNRL Anand Swaroop 10 8/3/23 AV2 CMV hNRL_AS321_s50T(2nd Prep) OGTC OGTC Anand Swaroop 11 8/3/23 AAV2 CMV hNRL AS321_L160p OGTC OGTC NNRL Anand Swaroop 12 8/10/23 AAV2 CMV hNRL AS321_L160fs OGTC OGTC NNRL Anand Swaroop 13 8/10/23 AAV2 CMV hNRL AS321_R218fs OGTC OGTC NNRL Anand Swaroop 14 8/25/23 AAV2 CamKIIa NES jRGECo1aWPRE SV40 OGTC OGTC NINDS Yi Gu 15 8/25/23 AAV5 VMD2 Tyr Dr. Brooks lab Brian Brooks lab Brian Brooks 16 8/25/23 AAV5 VMD2 EGFP Dr. Brooks lab Dr. Brooks lab Brian Brooks 17 8/28/23 AAV1 Zac2.1gfaABC1D-ick-GCaMP6f OGTC NINDS Yi Gu 18 8/28/23 AAV1 Zac2.1gfaABC1D-CYTO-GCaMP6f OGTC NINDS Yi Gu 19 8/28/23 AAV9 hM3Dq NINDS NINDS NINDS David Talmage 2. We have completed a study of CRISPR mediated gene therapy in a mouse model of retinal degeneration. Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration. This is a form of RP that is clinically highly significant because its prevalence and severity of disease. Currently there are several clinical trials of AAV-mediated gene replacement therapy ongoing with uncertain outcomes. As a potential alternative strategy to replacement gene therapy which are plagued by several unresolved issues, we sought to correct the mutation in situ via a simplified version of genome editing mediated by CRISPR. The rationale of the design takes advantage of the fact that a majority of RPGR disease causing mutations are frameshift alleles in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. While this falls short of complete rescue of all cells, it would still deliver meaningful alleviation of the disease and may significantly improve patients quality of life if successfully translated to the clinic. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCAs9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo. We have now expanded the study into human retina using iPSC derived retinal organoids derived from patients. We have successfully produced retinal organoids from patients iPSCs and are in the process of testing CRISPR mediated genome editing in an attempt to restore full-length RPGR-ORF15 expression and rescue the disease phenotype. Further, in collaboration with GEC headed by Dr. Lijin Dong, we have reprogrammed rd9 mouse cells into iPSCs and are in the process of generating retinal organoids. With the mouse retinal organoids, we will test ways to increase the efficiency of the rescue. Based on data from in vivo rd9 mouse studies, we have proposed a hypothesis that microhomology mediated recombination, rather than non-homologous end joining, is a major mechanism that drives the repair, giving the highly repetitive nature of the DNA sequence in this region. Retinal organoids carrying the rd9 mouse mutation will afford us the opportunity to study the mechanism in detail, and apply any new insight thus gained in the human retina. 3. We aim to provide investigators with retinal organoids at different ages as a testing platform of vector performance and correct gene expression in lieu of animals. We further advocate the use of organoids in many therapy studies to displace or reduce the use of animal models. To advance this goal, it is important that retinal organoid production be simplified, cost-effective and reproducible. We have continued methodological improvements in the process of retinal organoid generation from mouse and human iPSCs. Notably we have established a novel protocol which does not require a embryoid body generation step. We have tested it on 9 different iPSC lines and shown this method to be efficient, reproducible and shortens the production timeline by 10 days. It requires less specialty skills and should make the organoids model more widely available to investigators. A manuscript on this study will soon be submitted. 4. Gene therapy for USH1C in human retinal organoids. We have generated human retinal organoids derived from patients with USH1C mutations. We have demonstrated a cell biological disease phenotype in organoids older than 300 days. We have designed an AAV vector carrying a wt human USH1C gene and will deliver the vector into retinal organoids in culture. Detailed cell biological analyses will follow. These efforts should help us understand the disease pathophysiology as well as provide a prototype vector design for future clinical investigations for USH1C disease.