Susan L. Semple-Rowland - US grants

The long-term objective of the PI's research program is to identify and understand the biochemical cause or causes of retinal photoreceptor cell dysfunction. Such information would be of profound medical significance and may provide insight into the biomedical changes which underlie loss of retinal function in diseases of the human retina. Analyses of retinas of animals possessing genetic mutations which compromise photoreceptor cell function and viability provide an expedient means for accomplishing this goal. The proposed studies will focus on identifying and understanding protein changes associated with photoreceptor dysfunction in retinas of rd (retinal degeneration) chicks. Studies of the rd chick retina provide a unique opportunity to investigate biochemical changes underlying photoreceptor cell dysfunction because total loss visual cell function in the retinas of these animals occurs in the absence of gross developmental abnormalities and precedes cellular degeneration. The primary biochemical site(s) for the rd mutation is not known. Preliminary two-dimensional gel electrophoretic analyses of proteins extracted from homozygous rd [rd/rd], heterozygous carrier [+/rd] and normal [+/+] retina during development and prior to the appearance of degeneration have revealed two groups of proteins whose expression is affected by the mutation. The proposed experiments will provide information concerning the biochemical identities of these proteins, their tissue-specificity and their cellular and subcellular locations within the tissues which produce them. Amino acid analyses, peptide maps and N- terminal or internal peptide amino acid sequences of these proteins will be used to determine the degree of homology between the proteins. Monospecific antisera raised against these proteins will be used to identify the cells which produce the proteins and their intracellular location. These data along with the amino acid sequence data will provide important clues regarding the cellular functions of these proteins. Together, these results will improve our understanding the biochemical changes underlying photoreceptor dysfunction in the rd chick retina and may provide insight into general biochemical processes which cause loss of photoreceptor function in retinal disease.

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research program is to understand the biochemical processes in photoreceptor cells that if disrupted, lead to cell dysfunction and degeneration. The model system studied in this program is the retinal degeneration (rd) chicken, the only animal model for inherited retinal disease that possesses a cone-dominant retina. In the previous funding period, the investigators determined that the rd gene encoded photoreceptor guanylate cyclase 1 (GC1), a gene critical for cone and rod phototransduction. The results of their analyses show that the rd chicken is a model for Leber's congenital amaurosis (LCA1), an inherited retinal disease of the retinitis pigmentosa family of retinal degenerations that causes blindness in newborn infants. They propose that the GC1 null mutation in the rd chicken leads to abnormally low levels of cGMP in cone and rod cells, loss of phototransduction, and eventually to photoreceptor degeneration. The goal of the studies outlined in this proposal is to rescue the retinal degeneration phenotype of the rd chicken using somatic gene therapy. They will test the hypothesis that expression of normal GC1 in rd photoreceptor cells is sufficient to restore photoreceptor function and prevent photoreceptor cell death. The aims of this proposal are: (1) to identify fragments of the GCAP1 promoter that are capable of directing expression of reporter genes to photoreceptors in vitro (embryonic chicken retina cell culture) and in vivo (chicken embryos); (2) to examine the ability of selected GCAP1 promoters to drive expression of GC1 in photoreceptor cells in rd/rd retinal cultures; and (3) to rescue the retinal degeneration phenotype in the rd chicken using lentivirus to deliver a GCAP1/GC1 transgene to retinal progenitor or post-mitotic retinal cells. They will determine the transcription start point of GCAP1 and examine the specificity and activity levels of GCAP1 promoter fragments by transient transfection of chicken retinal cell cultures and by viral transduction of stage 9-11 chicken embryos. The ability of the GCAP1 promoter to drive GC1 expression in transiently-transfected rd/rd retinal cultures will be examined by measuring GC1 activity. To assess the effectiveness of the GC1 gene therapy, they will examine (1) the electroretinographic responses of the rd/rd retina, (2) retinal morphology, (3) GC1 expression, and (4) expression profiles of circadian-regulated genes whose normal temporal expression pattern is disrupted in rd/rd retina. Comparisons of rd/rd chickens treated during embryonic development versus at hatching will allow one to determine if the efficacy of the therapy is enhanced if it is administered to the earliest expression of the normal gene in vivo.

DESCRIPTION (provided by applicant): The goal of our research program is to develop therapies for inherited retinal diseases that support long-term photoreceptor function and survival. Three approaches have dominated efforts to develop effective for these diseases: corrective gene, anti-apoptotic, and neurotrophic therapies. The benefits obtained from these therapies have been relatively short-lived, many slowing but not preventing degeneration of the photoreceptor cells. The research outlined in this proposal is designed to determine if the therapeutic benefits of highly targeted combination treatments pairing corrective gene therapy with either anti-apoptotic or neurotrophic therapy are significantly greater than those obtained using corrective gene therapy alone. We will conduct these experiments in the cone-rich retinas of the GUCY1*B model of LCA1, a model system that is not only an excellent model for LCA1 but also provides a unique opportunity to examine the effects of combination treatments on cone cells. This project is divided into two research aims: AIM 1 - Develop dual-promoter, self inactivating, insulated lentiviral vectors capable of delivering multiple therapeutic transgenes to specific retinal cell types; AIM 2 -To test the hypothesis that degeneration of photoreceptors that have been treated with targeted corrective gene therapy can be prevented by combining corrective gene therapy with either targeted anti-apoptotic or targeted neurotrophic therapies. For Aim 1, dual-promoter vectors will be constructed that restrict expression of their therapeutic cargos to (1) cone and rod cells, (2) rod cells alone, and (3) M¿ller cells using cell-specific promoters. Vectors with the desired cellular expression characteristics will be modified to deliver corrective (RetGC1), anti-apoptotic (X-linked inhibitor of apoptosis, XIAP), and neurotrophic (rod derived cone viability factor, RdCVF; glial-derived neurotrophic factor, GDNF) therapies. Each of the therapeutic proteins, or the vectors carrying them, will be tagged with a unique fluorescent protein that we will use to analyze the effects of these treatments on photoreceptor cell survival. The vectors will be packaged into lentivirus and their expression characteristics will be analyzed in vivo using fluorescent microscopy, immuno-histochemistry, RT-PCR and western blot. For Aim 2, combination treatments pairing RetGC1 with either XIAP, RdCVF, or GDNF will be delivered to the retinas of GUCY1*B animals and the ability of these treatments to restore function to and promoter survival of photoreceptors will be evaluated using visual behavior assays, electroretinography, fluorescent microscopic analyses of retinal whole mounts, and molecular and protein analyses techniques. The fluorescent photomontages of whole mounts of the treated retinas will be analyzed using standard cell count methods and nearest neighbor and spatial clustering analyses techniques. The results of all of these analyses will permit us to determine if the combination treatments are effective in increasing survival of photoreceptor cells and if there is evidence of synergy between the delivered therapies that enhances photoreceptor survival. PUBLIC HEALTH RELEVANCE: Inherited retinal diseases that affect cone cells are among the most debilitating human retinal diseases. The research described in this proposal is designed to examine the ability of new combination treatments consisting of corrective gene, anti-apoptotic, and neurotrophic therapies to support and prevent degeneration of cone and rod cells affected by inherited retinal disease. This research will be carried out using an animal model of inherited retinal disease that possesses a cone-rich retina.