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，遗传复杂，丧失2-3Mb的功能 父系表达的印记基因的区域。编码2的多顺反子SNURF-Snrpn-snoRNA基因座 蛋白质和5个SnoRNAs，包括SNORD116作为一个主要基因，在决定表型方面起着重要作用。 PWS的治疗使用早期临床和分子诊断，结合行为调整、生长激素、 或正在进行的影响吞噬或行为的药物试验，但受限于对 多效性表型。尽管这些措施改善了结果，但都不是根治的，也不是有意义的临床治疗。 发病率仍然存在。表观遗传学或基因疗法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，涉及将PWS遗传元素微型化到单个 治疗性腺相关病毒载体。新的AAV载体系列将评估替代 80%的PWS基因，帮助确定基因丢失如何以及哪些基因丢失导致细胞和生化 PWS的表型。目标1检验假设，即AAV载体中的PWS-miniges将适当地 在细胞中表达PWS基因产物。PWS-Miniges包括SNURF-Snrpn在内源 带有多达5个snoRNA的双顺反子布局，以及在AAV反向末端之间的NDN-顺反子重复。使用 人在啮齿动物细胞中的基因序列将区分外源和内源基因座的表达。 另一种载体结构将编码SNURF-Snrpn内含子中的单个或聚集性snoRNA，或TO 在3‘内含子中模仿内源性组织。缩小AAV载体大小，并模仿内源性 调节，将使用具有广泛细胞特异性表达的合成NRF1基序阵列微型启动子， PWS-miniges的所有编码内容都在AAV包装能力内。Aim 2测试了这样的假设 AAV-mini基因可挽救PWS细胞表型。PWSβ-CELL(INS-1)模型显示了一个细胞自治 胰岛素分泌缺陷及多种激素和内质网的RNA和蛋白质产生缺陷 伴侣，源于一个或多个SNurf-Snrpn-snoRNA基因的丢失。AAV-PWS微型发电机 包装成AAV6血清型(最适合啮齿动物内分泌细胞)的载体将被转导到对照和 Pwsβ-细胞系(各3个)，评估胰岛素分泌水平、 多肽激素(如胰岛素、IAPP)和内质网伴侣蛋白(如HSPA5、HSP90B1)。目标3(体内 验证了AAV-miniges可以挽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致死性。急性呼吸道合并症患者注射AAV的疗效 将比较子宫和出生后第一天的情况。单个AAV载体中基因成分的小型化 不仅是PWS的体内基因治疗，而且是更广泛的疾病的体内基因治疗的绝佳机会。

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.