Jonathan Stoddard

Rhesus monkeys and humans share common susceptibility genes for age-related macular disease

Age-related macular degeneration (AMD), a complex multigenic disorder and the most common cause of vision loss in the elderly, is associated with polymorphisms in the LOC387715/ARMS2 and HTRA1 genes on 10q26. Like humans, macaque monkeys possess a macula and develop age-related macular pathologies including drusen, the phenotypic hallmark of AMD. We genotyped a cohort of 137 unrelated rhesus macaques with and without macular drusen. As in humans, one variant within LOC387715/ARMS2 and one in HTRA1 were significantly associated with affected status. HTRA1 and the predicted LOC387715/ARMS2 gene were both transcribed in rhesus and human retina and retinal pigment epithelium. Among several primate species, orthologous exons for the human LOC387715/ARMS2 gene were present only in Old World monkeys and apes. In functional analyses, the disease-associated HTRA1 polymorphism resulted in a 2-fold increase in gene expression, supporting a role in pathogenesis. These results demonstrate that two genes associated with AMD in humans are also associated with macular disease in rhesus macaques and that one of these genes is specific to higher primates. This is the first evidence that humans and macaques share the same genetic susceptibility factors for a common complex disease.

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation

Purpose Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. Methods Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. Results OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. Conclusions OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. Translational Relevance Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

Cell transplantation for retinal degeneration: Transition from rodent to nonhuman primate models

Transplantation of potentially therapeutic cells into the subretinal space is a promising prospective therapy for the treatment of retinal degenerative diseases including age-related macular degeneration (AMD). In rodent models with photoreceptor degeneration, subretinal transplantation of cell suspensions has repeatedly been demonstrated to rescue behaviorally measured vision, maintain electrophysiological responses from the retina and the brain, and slow the degeneration of rod and cone photoreceptors for extended periods. These studies have led to the initiation of a number of FDA-approved clinical trials for application of cell-based therapy for AMD and other retinal degenerative diseases. However, translation from rodent models directly into human clinical trials skips an important intermediary preclinical step that is needed to address critical issues for intraocular cell transplantation. These include determination of the most appropriate and least problematic surgical approach, the application of treatment in an eye with similar size and structure including the presence of a macula, and a thorough understanding of the immunological considerations regarding graft survival and the consequences of grafted cell rejection. This chapter will review these and related issues and will document current efforts to address these concerns.

Discovery of a CLN7 model of Batten disease in non-human primates

We have identified a natural Japanese macaque model of the childhood neurodegenerative disorder neuronal ceroid lipofuscinosis, commonly known as Batten Disease, caused by a homozygous frameshift mutation in the CLN7 gene (CLN7−/−). Affected macaques display progressive neurological deficits including visual impairment, tremor, incoordination, ataxia and impaired balance. Imaging, functional and pathological studies revealed that CLN7−/− macaques have reduced retinal thickness and retinal function early in disease, followed by profound cerebral and cerebellar atrophy that progresses over a five to six-year disease course. Histological analyses showed an accumulation of cerebral, cerebellar and cardiac storage material as well as degeneration of neurons, white matter fragmentation and reactive gliosis throughout the brain of affected animals. This novel CLN7−/− macaque model recapitulates key behavioral and neuropathological features of human Batten Disease and provides novel insights into the pathophysiology linked to CLN7 mutations. These animals will be invaluable for evaluating promising therapeutic strategies for this devastating disease.

Allogeneic iPSC-Derived RPE Cell Graft Failure Following Transplantation Into the Subretinal Space in Nonhuman Primates

Purpose To characterize the intraocular immune response following transplantation of iPS-derived allogeneic RPE cells into the subretinal space of non–immune-suppressed rhesus macaques. Methods GFP-labeled allogeneic iPS-derived RPE cells were transplanted into the subretinal space of one eye (n = 6), and into the contralateral eye 1 day to 4 weeks later, using a two-stage transretinal and transscleral approach. Retinas were examined pre- and post-surgery by color fundus photography, fundus autofluorescence, and optical coherence tomography (OCT) imaging. Animals were euthanized between 2 hours and 7 weeks following transplantation. T-cell (CD3), B-cell (CD20), and microglial (Iba1) responses were assessed immunohistochemically. Results Cells were delivered into the subretinal space in all eyes without leakage into the vitreous. Transplanted RPE cells were clearly visible at 4 days after surgery but were no longer detectable by 3 weeks. In localized areas within the bleb containing transplanted cells, T- and B-cell infiltrates and microglia were observed in the subretinal space and underlying choroid. A T-cell response predominated at 4 days, but converted to a B-cell response at 3 weeks. By 7 weeks, few infiltrates or microglia remained. Host RPE and choroid were disrupted in the immediate vicinity of the graft, with fibrosis in the subretinal space. Conclusions Engraftment of allogeneic RPE cells failed following transplantation into the subretinal space of rhesus macaques, likely due to rejection by the immune system. These data underscore the need for autologous cell sources and/or confirmation of adequate immune suppression to ensure survival of transplanted RPE cells.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)–based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Lebers hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.