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

Prader-Willi综合征（PWS）是一种多系统疾病，其遗传复杂，伴有2-3 Mb的功能丧失。 父系表达的印记基因的结构域。多顺反子SNURF-SNRPN-snoRNA基因座编码2 SNORD 116是决定表型的主要基因。 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， 或正在进行的药物试验，影响食欲过盛或行为，但由于对 多效性表型虽然这些措施改善了结果，但没有一个是治愈的， 发病率仍然存在。表观遗传学或遗传学治疗的潜在方法，以重新激活或取代沉默或 缺失的PWS基因由于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的方法，包括将PWS遗传元件小型化为单个 治疗性腺相关病毒（AAV）载体。新的AAV载体系列将评估取代 80%的PWS基因，有助于确定如何以及哪些基因丢失导致细胞和生化 PWS中的表型。目的1测试AAV载体中的PWS-小基因将适当地表达的假设。 在细胞中表达PWS基因产物。PWS-小基因包括内源基因中的SNURF-SNRPN。 在AAV反向末端重复序列之间具有多达5个snoRNA和NDN-顺反子的双顺反子布局。使用 啮齿动物细胞中的人基因序列将区分外源和内源基因座的表达。 替代的载体结构将在SNURF-SNRPN内含子中编码单个或成簇的snoRNA，或者， 在3'内含子中模仿内源组织。为了减小AAV载体大小，并模拟内源性 调控时，将使用对表达具有广泛细胞特异性的合成NRF 1基序阵列微型启动子， 在AAV包装容量内具有PWS-小基因的所有编码内容物。目标2检验假设， AAV-小基因可以拯救PWS细胞表型。PWS β-细胞（INS-1）模型显示了细胞自主性， 胰岛素分泌缺陷和多种激素和ER的RNA和蛋白质产生缺陷 分子伴侣，其来源于一个或多个Snurf-Snrpn-snoRNA基因的缺失。AAV-PWS小基因 包装为血清型AAV 6（最适合啮齿动物内分泌细胞）的载体将被转导到对照中， PWS β-细胞系（各3个），评估胰岛素分泌水平、 肽激素（例如，胰岛素，IAPP）和ER分子伴侣（例如，HSPA5、HSP90B1）。目标3（体内 翻译）测试了AAV-小基因可以拯救胰岛表型和新生儿胰岛细胞的假设。 缺乏所有PWS基因表达的小鼠模型的致死率。AAV注射在糖尿病患者中的治疗结果 比较子宫内与出生后第1天。单个AAV载体中遗传组分的小型化 代表了不仅PWS而且更广泛地在疾病中的体内基因治疗的显著机会。

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.