Prader-Willi syndrome (PWS) gene-domain and AAV miniaturization for gene therapy

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A multisystem disorder, Prader Willi syndrome (PWS) is genetically complex with loss of function of a 2-3 Mb domain of paternally-expressed imprinted genes. The polycistronic SNURF-SNRPN-snoRNA locus encoding 2 proteins and 5 snoRNAs, including SNORD116 as one major gene, is important in determining phenotype. Treatment in PWS uses early clinical and molecular diagnosis with behavioral modification, growth hormone, or ongoing drug trials that affect hyperphagia or behavior, but are limited by an incomplete understanding of pleiotropic phenotypes. Although these measures improve outcomes, none are a cure and significant clinical morbidity persists. Potential approaches for epigenetic or genetic therapy to reactivate or replace silent or missing PWS-genes are complicated by heterochromatic silencing and the many genes involved in disease. This project will test an innovative approach involving miniaturization of PWS genetic elements into a single therapeutic adeno-associated virus (AAV)-vector. The new AAV vector series will assess feasibility to replace 80% of PWS-genes, helping to determine how and which gene losses lead to cellular and biochemical phenotypes in PWS. Aim 1 tests the hypothesis that PWS-minigenes in an AAV-vector will appropriately express PWS gene products in cells. The PWS-minigenes include SNURF-SNRPN in the endogenous bicistronic layout with up to 5 snoRNAs, and the NDN-cistron, between AAV inverted terminal repeats. Use of human gene sequences in rodent cells will distinguish expression of exogenous and endogenous loci. Alternative vector structures will encode individual or clustered snoRNAs in SNURF-SNRPN introns or, to mimic the endogenous organization, in 3’ introns. To reduce AAV vector size, and mimic endogenous regulation, a synthetic NRF1-motif array mini-promoter having broad cell-specificity for expression will be used, with all coding content of PWS-minigenes within AAV packaging capacity. Aim 2 tests the hypothesis that AAV-minigenes can rescue PWS cellular phenotypes. A PWS β-cell (INS-1) model shows a cell-autonomous defect in insulin secretion and deficits in RNA and protein production of multiple hormones and ER chaperones, which derive from loss of one or more of the Snurf-Snrpn-snoRNA genes. AAV-PWS minigene vectors packaged as serotype AAV6 (optimal for rodent endocrine cells) will be transduced into control and PWS β-cell lines (3 each), assessing correction in levels of insulin secretion, mRNA and protein levels of peptide hormones (e.g., insulins, IAPP) and ER chaperones (e.g., HSPA5, HSP90B1). Aim 3 (in vivo translation) tests the hypothesis that AAV-minigenes can rescue the pancreatic islet phenotypes and neonatal lethality of a mouse model lacking expression of all PWS-genes. Therapeutic outcomes of AAV injections in utero vs. postnatal day 1 will be compared. Miniaturization of genetic components in a single AAV vector represents a remarkable opportunity for in vivo gene therapy of not only PWS but more broadly in disease.