Claudio Punzo - US grants

DESCRIPTION (provided by applicant): The inter-neuronal relationship between rod and cone photoreceptors in human and mouse is such that rod death always leads to cone death however; loss of cones has no effect on rods. This phenomenon plays an important role in the inherited retinal degenerative disease retinitis pigmentosa, as most disease-causing alleles identified encode for genes that are exclusively expressed in rods. Since cone death always follows rod death, and cones are essential for human vision, it is their loss that leads to blindness. We have recently proposed that cone death is a cell autonomous event caused by reduced nutrient uptake and showed that the insulin/AKT/mTOR pathway plays a crucial role during the periods of cone death. Systemic administration of insulin to retinitis pigmentosa mice prolongs cone survival. Here we propose to study how insulin prolongs cone survival. To that end we have now genetically activated the pathway in cones, by deletion of the phosphatase and tensin homolog (PTEN) and separately, by deletion of the tuberous sclerosis complex protein 1 (TSC1). Loss of PTEN or TSC1 further improves cone survival when compared to insulin administration, suggesting that genes downstream of PTEN and TSC1 have therapeutic potential to prolong vision in retinitis pigmentosa. Since loss of PTEN or TSC1 activates the kinases mechanistic target of rapamycin (mTOR) and AKT, genes that promote cone survival are predicted to be downstream of these two kinases. Because mTOR and AKT have hundreds of targets we will first delineate the contribution of these two kinases to cone survival seen upon loss of PTEN or TSC1. These experiments will be carried out in aims 1 & 2. Subsequently, in aim 3 we will use microarrays to identify target genes of mTOR and/or AKT that promote cone survival. Finally, we will tests the efficacy of such genes in vivo using recombinant Adeno-associated virus mediated gene transfer. Accomplishment of these aims will help design rational therapeutic approaches to extend vision in humans with retinitis pigmentosa.

PI: Claudio Punzo Project Summary The inter-neuronal relationship between rod and cone photoreceptors in human and mouse is such that rod death always leads to cone death; however, loss of cones has no effect on rods. This phenomenon plays an important role in the inherited retinal degenerative disease retinitis pigmentosa, as most disease-causing alleles identified encode for genes that are exclusively expressed in rods. Since cones are essential for human vision, it is their loss that leads to blindness. We have recently proposed that cone death is a cell autonomous event caused by reduced nutrient uptake, in particular glucose, and showed that cell autonomous activation of the kinase mammalian target of rapamycin complex 1 (mTORC1), by deletion of its negative regulator the tuberous sclerosis complex protein 1 (TSC1), significantly prolongs cone survival. Since our initial findings others have also supported the notion that secondary cone death in retinitis pigmentosa is manly caused by a shortage of glucose in cones. Our cell autonomous activation of mTORC1 in cones promoted cone survival by improving the following 3 glucose related processes: uptake, retention and metabolism. In this grant we want to test to which extent each of these 3 processes contributes to cone survival. We have identified 3 genes, through a rational analysis of our data, each representing one of these 3 processes. Here we propose to test how much each gene contributes to cone survival by rAAV mediated gene transfer to cones. We will test the cone survival effect mediated by each gene individually and in combination of two genes at the same time. To ensure that our approach is mutation independent we will carry out our experiments in two mouse models of retinitis pigmentosa. Accomplishment of the proposed research will lay the foundation for the design of a rational therapeutic approach to extend vision in humans with retinitis pigmentosa.

PROJECT SUMMARY Given the recent FDA approval of targeted AAV gene therapy platforms and of small-molecule splicing modulators as treatments for genetic neurological disorders, our goal is to apply these powerful technologies to prevent the progressive optic neuropathy and blindness that develops in patients with the genetic recessive disease, Familial dysautonomia (FD). FD results from a splice site mutation in intron 20 of the gene ELP1 (formerly called IKBKAP). As a consequence of the mis-splicing, exon 20 is variably skipped, the mutant mRNA degraded, resulting in reduced levels of the encoded protein, Elp1.. Interestingly, the ability to splice the mutated pre-mRNA varies according to tissue type, with neurons least capable of splicing the mutated pre- mRNA. While the majority of the clinical deficits are due to the devastation of the sensory and autonomic nervous systems, as patients enter their teens, their macular retinal ganglion cells progressively die, manifesting as visual loss. Mouse that are null for Elp1 are embryonic lethal so the field has, until now, taken two distinct strategies to generate mouse models to investigate FD: (i) generation of conditional knock-out mice (CKO) using cell-type specific cre-driven promoters; and (ii) transgenic mice that contain the human FD ELP1 splicing mutation. The former approach has generated mouse models that recapitulate the FD optic neuropathy that results from the progressive death of retinal ganglion cells. These mice are an excellent pre- clinical model for testing the effectiveness of gene therapy for preventing the progressive demise of retinal ganglion cells (Aim 1A). However this model does not lend itself to testing the effectiveness of splicing enhancer compounds since it lacks the FD splicing mutation. The latter approach has culminated in the generation of transgenic mice that include copies of the human FD ELP1 mutated gene. These mice are asymptomatic unless they are crossed to a hypomorph or null background mouse, but these compound mice are typically too sick to investigate consistently. Here we will make a new ?hybrid? line by crossing in the human FD ELP1 mutated gene into our retina-specific CKO line (Pax6-cre;Elp1flox/flox) to overcome these major challenges to the field. In so doing, we will generate a single mouse model that manifests the human FD optic neuropathy, in an otherwise healthy background, and contains the splice site mutation, which can be used to test a variety of therapeutic approaches (Aim1A, B). The overall aim of this proposal is to assess and compare two methods for restoring normal levels of the Elp1 protein in this new model mouse retinae using: (i) AAV2- mediated gene therapy (gene reintroduction) of the wild type Elp1 gene injected intravitreously, and (ii) a novel splicing enhancer compound that has been shown to promote the inclusion of exon 20 in the mutant FD gene in the retina, delivered orally through diet. Our goal is to test which method best mitigates the death of retinal ganglion cells in addition to interrogating whether a combination of both methods (Aim 1C) will have additive effects on promoting the survival of retinal ganglion cells, given they work via two distinct pathways.

PI: Claudio Punzo Project Summary Retinal-pigmented epithelium atrophy (RPE) that results in geographic atrophy (GA) in humans is one of the leading causes for blindness in the industrialized world. This is because there is currently no treatment available to prevent RPE atrophy and thus GA, which is an advanced form of Age-related Macular Degeneration (AMD). The disease is characterized by focal RPE cell loss. Because the RPE maintains photoreceptor homeostasis, photoreceptors die as well, which then leads to blindness. Recently, it has been recognized that the high metabolic demands of photoreceptors may contribute to disease progression in AMD, in particular, because photoreceptors and RPE metabolism are tightly linked. Two key findings imply photoreceptors in disease pathogenesis. First, the distribution of soft drusen and subretinal drusenoid deposits mirrors the distribution of cones and rods, respectively. This has led to the proposal that the metabolic needs of photoreceptors are what drives deposit formation. Second, macular translocation procedures, which were developed to save macular cones from dying RPE cells revealed that the new region where the cones where translocated redeveloped GA. Here it is thought that the high metabolic demands of cones are what causes RPE stress. However, whether photoreceptor metabolism differs between AMD patients and non-diseased individuals remained unclear. We recently showed that PRs of AMD patients display signs of nutrient derivation as they upregulate genes associated with an adaptive response to a glucose shortage. By mimicking this adaptive response in mouse photoreceptors we were able to induce a subset of pathologies that are reminiscent of those seen in humans with AMD, including focal RPE atrophy. The goal of this project is to identify what exactly causes the pathologies seen. We propose in aim 1 to further analyze our model and to determine how RPE cells die. Thereafter, in aim 2, we will dissect genetically the signaling pathway that we have used to manipulate photoreceptor metabolism in order to hone in on the metabolic changes that cause disease. Finally, in aim 3, we will use metabolomics, lipidomics and transcriptomics to identify the underlying gene expression changes that cause disease and test putative candidate mechanisms in vivo. Accomplishment of the proposed research will help understand how photoreceptors can cause RPE atrophy.