This laboratory is appropriately titled Translational Research, as we use inherited retinal degenerations identified in the clinic as both a source of clues about retinal function and dysfunction and a target for research in therapeutic intervention. We use normal rodents and rodents that are genetically altered to mimic human retinal disease to study the characteristics (phenotype), molecular genetics, physiological mechanisms and possible treatments of inherited retinal degenerations. Our laboratory applies the techniques of light and electron microscopy, immunohistochemistry, biochemistry, and molecular biology to human and animal retinal tissue, as well as the electroretinogram (ERG) and behavioral measurements to access retinal function in live animals. (1) X-linked juvenile retinoschisis (XLRS): Disease mechanisms and therapy development. XLRS is a leading cause of juvenile macular degeneration in human males. Our goal is to develop therapy for XLRS, including possibilities for human gene transfer therapy. Our current understanding is based on a study of human affected patients and on analysis of the XLRS mouse model which we developed in this laboratory. We have probed the biochemistry and sub-cellular localization of the RS protein and have localized RS to particular cell membrane sites of photoreceptors and synapses and measured changes in key membrane proteins in synapses. We discovered molecular interactions between RS and photoreceptor membrane phospholipids that may explain its role in neuronal structure and retinal signaling. We cloned and characterized the human gene promoter region and have identified the key regulatory sites. A detailed study of long-term disease progression in the XLRS mouse revealed significant correlations between degenerative structural changes and functional neuronal signaling abnormalities. Such studies currently are not possible in human and provide us better understanding of disease mechanisms and give clues on designing appropriate endpoint metrics for eventual human clinical trial. We showed retinal rescue of structure and function in the XLRS mouse by AAV vector RS gene transfer, and we are now characterizing appropriate intervention times in disease progression and parameters that lead to success or failure of gene transfer. All of this is in preparation to designing and conducting a human AAV-RS gene transfer experiment. (2) Animal models of retinal degenerations: Pathophysiology and therapy exploration. We have studied a number of mouse and rat models of human retinal degeneration diseases for purposes of elucidating the existence and mechanism of retinal neural signaling deficiencies leading to blindness. We developed a novel method for blocking specific membrane channels and receptors in the retina using antibodies. Using K-channel antibodies injected in vivo, we altered the electroretinogram (ERG)of the RCS rat retinal degeneration model in characteristic ways that implicate potassium channel activity in response generation. Simultaneously we could localize these antibodies to particular retinal cell types and subcellular locations. This approach provides a powerful opportunity to probe mechanisms of disease states using non-invasive ERG response recordings to probe pathophysiologic mechanisms in vivo, even during treatment. Retinal visual function depends on rod and cone photoreceptors and associated neural pathways. We are studying the cone pathway responses in mutant mice that lack functional rods due to genetic knockout of the rhodopsin gene but which still have an intact rod pathway. We have found evidence that the rod pathway affects the time course of light adaptation in the cone pathway; that rods without photoreceptive membranes still constitutively activate the rod pathway; and that when rods are replaced by cones (in a mouse genetic mutant moddel) the cones activate the normal rod pathway, which is a physiological demonstration of retinal plasticity. In addition to gene therapy for retinal degenerations due to specific genetic mutations, intervention is being explored using lens epithelial derived growth factor (LEDGF) and heat shock protein 27 (HSP27). These factors could be useful in therapy for a broad range of retinal degenerations. We cloned both genes into viral vectors for retinal delivery into the intact rodent eye. Both agents slowed the natural course of degeneration in the RCS rat model of MERTK genetic degeneration. Further analysis is underway to learn the concomitant changes in gene and protein expression in relation to the disease state. We have previously studied ciliary neurotrophic factor (CNTF) in animal disease models and have subsequently applied this to a human Phase I clinical trial for retinitis pigmentosa (RP) which we successfully completed and published in 2006. Subsequent study showed that exogenous CNTF downregulated expression of proteins in the photoreceptor transduction cascade, which provides clues as to therapeutic mechanism found in animals. We have developed a protocol for a Phase II human CNTF efficacy trial for macular degeneration and RP. (3) Photoreceptor plasticity and homeostasis in normal and disease retina. A critical facet of retinal neurodegenerative disease involves the structural changes, particularly to the photoreceptor outer segments (OS) that precede photoreceptor death, causing loss of vision. A critical question is whether the outer segment may exhibit sufficient structural plasticity to support elongation of OS that have been shortened by disease states and whether this would promote survival of the photoreceptor cell. The goal of the work is to investigate the molecules that are important in the regulation of OS length under light stress and genetic degenerative conditions. We are focusing on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including RAC1. Human Protocol 03-EI-0033. X-Linked Juvenile Retinoschisis - Clinical and Molecular Studies. (PI: P.A. Sieving). A genotype-phenotype study of XLRS which results in splitting of the retinal layers. A better understanding of XLRS development might lead to improved treatments through gene transfer. Human Protocol 03-EI-0179. Investigation of the Effect of Dietary Docosahexaenoic Acid (DHA) Supplementation on Macular Function in Subjects with Autosomal Dominant Stargardt-Like and Autosomal Recessive Stargardt Macular Dystrophy. (PI: P.A. Sieving). Human Protocol 03-EI-0234. A Phase I Study of NT-501-10 and NT-501-6A.02, Implants of Encapsulated Human NTC-210 Cells Releasing Ciliary Neurotrophic Factor (CNTF), in Patients with Retinitis Pigmentosa. (PI: P.A. Sieving). Evaluate safety of CNTF implant in the human eye of retinal degeneration subjects. CNTF protects against retinal degeneration in animal models. Study addresses a major treatment challenge of delivery directly into the human eye. Published: PNAS (2006) Ciliary neurotrophic factor (CNTF) for human retinal degeneration: Phase I trial of CNTF delivered by encapsulated cell intraocular implants. Human Protocol 03-EI-0255. Pilot Study on the Effect of Vitamin A Supplementation on Cone Function in Retinitis Pigmentosa (RP). (PI: P.A. Sieving). A protocol to evaluate whether high dose oral vitamin A will improve retinal function acutely in patients with RP. Human Protocol 06-EI-0071. Phase II Study of Implants of Encapsulated Human NTC-201 Cells Releasing Ciliary Neurotrophic Factor (CNTF), in Participants with Visual Acuity Impairment Associated with Atrophic Macular Degeneration (PI: P.A. Sieving). Evaluate the safety and effectiveness of ciliary neurotrophic factor (CNTF) implants on vision in participants with atrophic macular degeneration.