Protocols for Visually Guided Navigation Assessment of Efficacy of Retina-Directed Cell or Gene Therapy in Canines

There has been marked progress in recent years in developing gene delivery approaches for the treatment of inherited blinding diseases. Many of the proof-of-concept studies have utilized rodent models of retinal degeneration. In those models, tests of visual function include a modified water maze swim test, optokinetic nystagmus, and light-dark activity assays. Test paradigms used in rodents can be difficult to replicate in large animals due to their size and awareness of non-visual aspects of the test system. Two types of visual behavior assays have been utilized in canines: an obstacle avoidance course and a forced choice Y maze. Given the progress in developing cell and gene therapies in large animals, such tests will become more and more valuable. This study provides guidelines for carrying out such tests and assesses the challenges and benefits associated with each test

Use of induced pluripotent stem cell models to probe the pathogenesis of Choroideremia and to develop a potential treatment

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 β-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. Keywords: Choroideremia, Human iPSCs, Retinal pigmented epithelium, Gene therapy, Adeno-associated virus, Phagocytosis, Prenylation, REP

RNA Interference-Based Therapy for Spinocerebellar Ataxia Type 7 Retinal Degeneration

<div><p>Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by loss of motor coordination and retinal degeneration with no current therapies in the clinic. The causative mutation is an expanded CAG repeat in the ataxin-7 gene whose mutant protein product causes cerebellar and brainstem degeneration and retinal cone-rod dystrophy. Here, we reduced the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina by RNA interference and evaluated retinal function 23 weeks post injection. We observed a preservation of normal retinal function and no adverse toxicity with ≥50% reduction of mutant and wildtype ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 retinal therapy.</p></div

Assessing toxicity post-injection at 30 weeks.

<p>(a) Hematoxylin and eosin staining of retinas at 30 weeks (n = 3 per group) (b) Optical coherence tomography (OCT) images of the retinas at the region of the optic nerve (n≥6 per group). (c) Retinal thickness measured using the OCT images. (d) Relative GFAP mRNA levels in the injected retinas by RT-qPCR analysis. For (c; n≥6 per group) and (d; n = 3 per group), results are represented as mean ±SEM.</p

Retinal function 30 weeks post-injection.

<p>(a) ERG b-wave of the mixed rod-cone response, n≥4 (b) ERG a-wave of the mixed rod-cone response, n≥4 (c) 5 Hz flicker (cone) response, n≥4 (d) Optokinetic response measured by the spatial frequency, n≥3. Results are represented as mean ±SEM, *p<0.05.</p

Reduction of ataxin-7 mRNA in the SCA7 mouse retina.

<p>(a) Relative levels of human (top panel) or mouse (bottom panel) ataxin-7 mRNA levels one month post-injection with AAV2/1 miC or miS4. (b) Representative western blot demonstrating mutant human (upper band, denoted by arrow) and mouse ataxin-7 (lower band) protein levels one month post injection of AAV2/1 miC or miS4. For quantification (c), ataxin-7 protein levels were normalized to β-actin (upper panel, human protein; lower panel, mouse protein). For all panels, results are represented as mean ±SEM (n = 3), ***p<0.001, **P<0.01,*p<0.05.</p