Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity.

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by Mertk loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of-Mertk in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy.SIGNIFICANCE STATEMENT Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking Mertk), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of Mertk function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness.

The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.

Systemic rapamycin inhibits retinal and choroidal neovascularization in mice.

PURPOSE: Rapamycin exhibits significant antitumor/antiangiogenic activity that is coupled with a decrease in vascular endothelial growth factor (VEGF) production and a reduction in the response of vascular endothelial cells to stimulation by VEGF. VEGF plays a significant role in neovascular pathologies of the eye, thus we tested the possibility of using rapamycin to inhibit retinal and choroidal neovascularization (CNV). METHODS: CNV was induced in adult mice with laser photocoagulation. Retinal neovascularization was induced using the retinopathy of prematurity (ROP) hyperoxia/hypoxia model. Experimental animals received intraperitoneal (ip) injections of rapamycin (2 mg/kg/day or 4 mg/kg/day) for 1-2 weeks. Controls were not treated or received ip injections of phosphate buffered saline (PBS). Eyes were analyzed histologically for evidence of CNV or retinal neovascularization. ROP eyes were further analyzed for changes in VEGF and VEGF receptor (Flt-1 and Flk-1) protein content following rapamycin treatment. RESULTS: Rapamycin significantly reduced the extent of neovascularization in both the CNV and the ROP model. Immunohistochemical staining of treated and untreated ROP retina did not reveal a significant reduction in levels of VEGF protein or its receptors. Immunostaining for Flt-1 increased, while no obvious changes in Flk-1 were observed. Quantitative analysis of total protein via enzyme linked immunosorbent assay (ELISA) confirmed an increase in Flt-1 and VEGF, following drug treatment, with no effect on Flk-1. CONCLUSIONS: These results suggest rapamycin may provide an effective new treatment for ocular neovascularization.

In utero gene therapy rescues vision in a murine model of congenital blindness.

The congenital retinal blindness known as Leber congenital amaurosis (LCA) can be caused by mutations in the RPE65 gene. RPE65 plays a critical role in the visual cycle that produces the photosensitive pigment rhodopsin. Recent evidence from human studies of LCA indicates that earlier rather than later intervention may be more likely to restore vision. We determined the impact of in utero delivery of the human RPE65 cDNA to retinal pigment epithelium cells in a murine model of LCA, the Rpe65(-/-) mouse, using a serotype 2 adeno-associated virus packaged within an AAV1 capsid (AAV2/1). Delivery of AAV2/1-CMV-hRPE65 to fetuses (embryonic day 14) resulted in efficient transduction of retinal pigment epithelium, restoration of visual function, and measurable rhodopsin. The results demonstrate AAV-mediated correction of the deficit and suggest that in utero retinal gene delivery may be a useful approach for treating a variety of blinding congenital retinal diseases.

Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity.

PURPOSE: Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. METHODS: Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. RESULTS: AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. CONCLUSION: AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

Gene therapy and animal models for retinal disease.

Those plagued by retinal diseases are often robbed of their vision, as often, effective treatments do not exist. Knowledge of the pathophysiology of retinal diseases stems from research on available animal models. Gene therapy may be useful for both genetic and acquired retinal diseases. This review will focus on retinal diseases for which gene therapy has demonstrated promise. The diseases are presented in order of the age at which they are generally first symptomatic and include retinopathy of prematurity, Leber congenital amaurosis, mucopolysaccharidoses, retinoblastoma, retinitis pigmentosa, diabetic retinopathy, glaucoma and age-related macular degeneration. We will describe the animal models used to study these disorders and emphasize the progress that has been made in using gene therapy for the treatment of retinal disease.

A deviant immune response to viral proteins and transgene product is generated on subretinal administration of adenovirus and adeno-associated virus.

The immune response after ocular exposure to foreign antigens varies substantially from that of a typical systemic response. Anterior chamber associated immune deviation (ACAID) has been well documented. The immune response of the subretinal space has not been studied in as much detail. Here, we characterized the immune response of the subretinal space when it encounters the antigens AdV-GFP and AAV-GFP (recombinant adenovirus or adeno-associated virus, respectively), each delivering the reporter gene encoding green fluorescent protein (GFP). Results indicate that the subretinal space possesses an immune-deviant property similar to ACAID. AdV-elicited immune responses following subretinal injections are significantly reduced compared with systemic responses elicited by intradermal injections of the same virus. Furthermore, subretinal AdV administration results in transduction of retinal pigment epithelial cells (RPE), which are the potential antigen presenting cells of the retina. This subsequently generates a population of immunosuppressive Th2-type, cytokine-secreting, splenic T cells. This response may be advantageous to the development of ocular gene therapy.