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

Prader-Willi 综合征 (PWS) 基因域和 AAV 小型化用于基因治疗

基本信息

批准号：
10593218
负责人：
Robert D Nicholls
金额：
$ 22.95万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593218
关键词：
Affect Animal Model Architecture Behavior Behavior Therapy Behavioral Beta Cell Biochemical Biological Assay Biological Models Blood Glucose Cell Line Cell model Cells Cistrons Clinical Code Cognitive Complex Data Defect Dependovirus Development Disease Endocrine Endoplasmic Reticulum Etiology Failure to Thrive Family suidae Foundations GRP78 gene Gene Transduction Agent Genes Genetic Glucagon Hormones Human Hyperphagia Hypoglycemia Immunohistochemistry In Vitro Individual Infection Injections Insulin Introns Inverted Terminal Repeat Islets of Langerhans Lead Measures Messenger RNA Miniaturization Modeling Molecular Chaperones Molecular Diagnosis Morbid Obesity Morbidity - disease rate Mus Muscle hypotonia Neonatal Pharmaceutical Preparations Phenotype Play Prader-Willi Syndrome Preclinical Testing Problem behavior Production Protein Secretion Proteins RNA Regulation Regulatory Element Rodent Role SNRPN Series Serotyping Small Nucleolar RNA Somatotropin Specificity Structure System Testing Therapeutic Transfection Translations Viral Genes Viral Genome Viral Packaging Viral Vector Western Blotting adeno-associated viral vector cellular transduction clinical diagnosis clinical phenotype design digital drug testing endocrine pancreas development epigenetic therapy expression vector fetal gene product gene therapy genetic element genome editing imprint improved outcome in utero in vivo innovation insulin secretion insulinoma islet loss of function miniaturize mouse model novel novel strategies novel therapeutics peptide hormone postnatal promoter protein expression symptom management therapeutic evaluation therapy outcome vector

项目摘要

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.

普拉德威利综合征 (PWS) 是一种多系统疾病，其基因复杂，具有 2-3 Mb 功能丧失父系表达的印记基因的结构域，编码 2 的多顺反子 SNURF-SNRPN-snoRNA 基因座。蛋白质和 5 种 snoRNA，包括作为主要基因的 SNORD116，对于确定表型很重要。 PWS 的治疗采用早期临床和分子诊断，包括行为矫正、生长激素、或正在进行的影响食欲过盛或行为的药物试验，但由于不完全了解而受到限制尽管这些措施可以改善结果，但都不是治愈方法且具有显着的临床意义。表观遗传或基因治疗的潜在方法仍然存在，以重新激活或取代沉默或替代。缺失的 PWS 基因因异染色质沉默和许多与疾病有关的基因而变得复杂。该项目将测试一种创新方法，将 PWS 遗传元件小型化为单个新的 AAV 载体系列将评估替代治疗性腺相关病毒 (AAV) 的可行性。 80% 的 PWS 基因，有助于确定基因丢失如何以及哪些基因丢失导致细胞和生化目标 1 测试 AAV 载体中的 PWS 小基因会适当地发挥作用的假设。在细胞中表达PWS基因产物，PWS小基因包括内源性的SNURF-SNRPN。使用最多 5 个 snoRNA 和 NDN 顺反子的双顺反子布局，在 AAV 反向末端重复序列之间使用。啮齿动物细胞中的人类基因序列将区分外源和内源基因座的表达。替代载体结构将编码 SNURF-SNRPN 内含子中的单个或簇状 snoRNA，或者，模仿内源性组织，在 3' 内含子中减少 AAV 载体大小，并模仿内源性。调节，将使用具有广泛细胞表达特异性的合成 NRF1 基序阵列微型启动子，目标 2 测试了 AAV 包装容量内 PWS-小基因的所有编码内容。 AAV-小基因可以拯救 PWS 细胞表型 PWS β 细胞 (INS-1) 模型显示出细胞自主性。胰岛素分泌缺陷以及多种激素和 ER 的 RNA 和蛋白质产生缺陷分子伴侣，源自一个或多个 Surf-Snrpn-snoRNA 基因的丢失。包装为血清型 AAV6（最适合啮齿动物内分泌细胞）的载体将被转导至对照和 PWS β 细胞系（各 3 个），评估胰岛素分泌水平、mRNA 和蛋白质水平的校正肽激素（例如胰岛素、IAPP）和 ER 伴侣（例如 HSPA5、HSP90B1）Aim 3（体内）。翻译）检验了 AAV 小基因可以拯救胰岛表型和新生儿的假设缺乏所有 PWS 基因表达的小鼠模型的 AAV 注射治疗结果。将比较子宫内与出生后第 1 天单个 AAV 载体中遗传成分的小型化。这不仅为 PWS 的体内基因治疗提供了绝佳的机会，而且还为更广泛的疾病提供了绝佳的机会。