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.

多系统疾病，Prader Willi综合征（PWS）在遗传上很复杂，功能丧失2-3 MB 亲子表达的印迹基因的结构域。编码2 蛋白质和5个snornas，包括SNORD116作为一个主要基因，对于确定表型很重要。 PWS中的治疗使用早期临床和分子诊断进行行为修饰，生长马酮，或正在进行的影响脾气或行为的药物试验，但受到对多效性表型。尽管这些措施改善了预后，但没有一种治愈和明显的临床发病率仍然存在。表观遗传学或基因治疗的潜在方法重新激活或替换无声或杂质沉默和疾病中的许多基因使缺失的PWS生成变得复杂。该项目将测试一种创新的方法，涉及将PWS遗传元素微型化为单个治疗性腺相关病毒（AAV） - 载体。新的AAV矢量系列将评估替换的可行性 80％的PWS基因，有助于确定如何以及哪些基因损失导致细胞和生化 PWS中的表型。 AIM 1检验了AAV-vector中的PWS小米的假设在细胞中表达PWS基因产物。 PWS小米包括内源性的Snurf-SnRPN Bicistronic布局，最多5个snornas和NDN-Cistron在AAV倒终端重复序列之间。使用啮齿动物细胞中的人类基因序列将区分外源性和内源性基因座的表达。替代矢量结构将编码SNRF-SNRPN inrons中的单个或聚类的snornas，或者在3'方法中模仿内源性组织。减少AAV矢量大小，并模仿内源性调节，将使用具有宽细胞特异性的合成NRF1-MOTIF阵列迷你启动器，用于表达，在AAV包装能力内，PWS微型烯的所有编码内容。 AIM 2检验了以下假设 AAV-Minigenes可以营救PWS细胞表型。 PWSβ细胞（INS-1）模型显示了细胞自主胰岛素分泌缺陷并定义了多种激素和ER的RNA和蛋白质产生伴侣，它源于损失一个或多个SNRF-SNRPN-SNORNA基因。 AAV-PWS小型包装为血清型AAV6（啮齿动物内分泌细胞的最佳）的载体将被翻译成对照和 PWSβ细胞系（每条3），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）。目标3（体内翻译）检验了AAV微型烯可以营救胰岛表型和新生儿的假设小鼠模型的致死性，缺乏所有PWS基因的表达。 AAV注射的治疗结果将比较子宫与产后第1天。单个AAV载体中遗传成分的微型化不仅是PW，而且在疾病中更广泛的体内基因治疗是一个了不起的机会。