Mechanisms, modeling and therapies of retinal and macular neurodegeneration

National Eye Institute

Investigators

Linked publications & trials

Abstract

Whole exome sequencing (WES) of patients with inherited retinal diseases (IRDs) Elucidating the genetic variations leading to disease development and progression is the first step to be able to provide patients with both accurate genetic counseling and development of therapeutic strategies. WES analyses of IRD patients have identified known and novel potentially disease-causing mutations in about 50% of our IRD cohort. Whole exome and whole genome sequencing of age-related macular degeneration (AMD) families We have generated whole exome and whole genome sequencing data for over 107 large multigenerational AMD families. Segregation of variants, case control collapsing analysis, linkage and literature based know gene analysis has been completed. We are now compiling the data for publication. By analyzing WES data from a cohort of AMD families, we have identified ultra-rare variants in complement factors 8A and 8B in four families and validated rare variants in several previously reported genes (e.g., TIMP3, C9 and CFH). Our studies further establish the role of complement pathway in AMD pathogenesis and suggest components of terminal MAC as candidates for therapeutic intervention. PILRB as a target gene for AMD AMD-associated eVariants regulate PILRB/PILRA locus, uncovering complement/immune pathways' role in disease progression. We evaluate which gene at this locus may directly contribute to retinal biology, we generated Pilra, Pilrb2 and Pilrb1/2 KO mice using CRISPR-Cas9 approach. Pilra and Pilrb2 KO mice did not show any obvious photoreceptor defect. Notably, Pilrb1/2 double KO mice show early but stationary photoreceptor dysfunction, revealing alteration in phototransduction cascade with no obvious change in photoreceptor number or phorphology. Notably, novel Pilr expression is detected in mouse photoreceptors, suggesting its potential significance as a key participant in phototransduction signaling and visual function. Further investigation are in progress and a manuscript is in preparation. Methylation quantitative trait loci (mQTLs) associated with AMD Previously, we profiled transcriptomes of the human peripheral retina samples and identified expression quantitative trait loci (eQTLs) including those relevant to AMD. To further evaluate how non-coding variants can contribute to AMD, we have performed array-based DNA methylation profiling of 152 postmortem retinas and identified 37,453 variants associated in cis (<1 Mb) with CpG sites known as methylation quantitative trait loci (mQTLs). Gene ontology analysis of mGenes identified genes involved in regulation of GTPase activity, actin filament and synaptic signaling. We used these genetic associations to infer potential causal relationships between gene expression and DNA methylation. Integrating AMD GWAS results with eQTL or mQTL using multiple colocalization methods revealed previously reported 9 GWAS loci including CFH, ARMS2/HTRA1, or LINC01004. Furthermore, integration of expression with methylation identified 13,747 expression quantitative trait methylation (eQTMs), and target genes of eQTM are enriched in oxidative phosphorylation, electron transport chain and translational initiation. The high correlation of genetic effects between methylation and gene expression highlights the potential to identify target genes in AMD for functional studies. Decoding the regulatory role of non-coding genome in retinal and macular degeneration Using the human macula transcriptome and eQTLs, we performed integration of AMD GWAS with eQTL proposed potential target genes and regulatory mechanisms at 6 AMD loci including ACAD10, RDH5/CD63 and PILRB/PILRA. We are performing colocalization analysis to identify novel genes and pathways associated with AMD. To further advance our understanding, we are performing Massively Parallel Reporter Assays that will characterize 3000 AMD eQTLs that overlap with regulatory elements, or are associated with AMD, Retnet, and Retina-enriched genes. We predict the identification and characterization of CREs regulating genes important for retinal function along with potential genetic variants that could compromise their function. Disease modeling and gene therapy of NPHP5-LCA Fibroblasts from 3 families affected by NPHP5-LCA were examined for cilia defects. We observed elongation of primary cilia in fibroblasts of two siblings carrying biallelic truncation mutations in NPHP5. Skin biopsies from these two patients and a familial control were used to derive iPSCs, which were differentiated into RPE cells and retinal organoids. Consistently, cilia extension phenotype and diminished CEP290 were also observed in patient RPE. Retinal organoids showed impaired outer segment development with mislocalization of opsins and other phototransduction proteins and loss of CEP290 protein. This phenotype was rescued by administration of an AAV vector encoding correct copy of NPHP5 gene, suggesting a potential future therapeutic avenue. Drug candidates for photoreceptor survival in retinal degenerative diseases We previously identified 5 drug candidates, including a lead candidate reserpine, which can sustain photoreceptors in mouse and human retinal organoids derived from iPSCs carrying mutations in the CEP290-LCA. Reserpine also showed efficacy in slowing down photoreceptor cell death in the rd16 mouse retina. Further investigations revealed misregulation of autophagic flux in patient stem cell-derived organoids and in the rd16 retina, resulting in downregulation of autophagy adaptor p62. Restoration of p62 level by specific autophagy inhibitors reduced the level of HDAC6 and improved cellular microtubule networks and biogenesis of the outer segment. We then evaluated the effectiveness of reserpine in rd10 mouse model of retinal degeneration using electrophysiological and histological methods, including vertical sections and flat-mounted retinas. Notably, reserpine was effective in maintaining photoreceptors for longer durations in rd10 retina. We have also examined neuroprotective effects of reserpine and other small molecules throughout the progression of retinal degeneration in the P23H animal model of retinitis pigmentosa. Electrophysiology, histological assessments using hematoxylin staining and immunohistochemistry on sections of rats treated with reserpine further confirm reserpine as a promising candidate drug for retinal degeneration therapy to prolong photoreceptor survival. Are any products or services commercially available or being developed that have arisen from the research in this project? Yes.