Rescue of multiorgan pathophysiology by adeno-associated virus 1 gene therapy

AAV载体作为基因治疗剂(3-5)具有很大的潜力，尤其是对囊性纤维化(CF)的治疗。研究 起源于我们的小组导致了rAAV在人类中的第一次使用(2)。许多研究表明，AAV载体 可安全使用(5，6，9)。然而，主要的挑战是，他们还没有实现可再生的Thera- 因此，有必要对甲型肝炎病毒的基因治疗采取新的途径。在以前的工作中，我们有 确定了三种缓解这些问题的新策略：1)使用AAV1，它对肺更具趋向性； 2)使用27-264，这是通过新机制校正∆F508的cftr的截断版本；以及3)包括 一只强大的鸡肌动蛋白(β)启动子的Sion。鉴于CF是一种多器官疾病，基于基因的治疗 必须针对几个器官，这些器官的细胞通常会翻转(3，4)，这使得使用病毒是有利的 如果要用于单一治疗，它可以转导多种细胞类型。为了进一步探索这一点 方法，我们将使用携带G551D突变的雪貂模型，该模型对CF增强剂VX-770有反应。 我们提出了三个总体具体目标： 目的1：评价AAV1基因治疗转导受感染器官的全身和呼吸道给药效果。 参见为了使基因疗法对慢性萎缩性胃炎有效，它必须纠正体内几个器官中CFTR功能的缺陷。 除了呼吸道以外的身体。这里讨论的问题是转导是否可以发生在外周器官中 在自然发生的AAV1的呼吸道输送之后。目的2：确定治疗的持续时间 含截短cftr基因的AAV1载体的作用CF基因治疗的首要问题是 基因转导能否挽救CF表型以及治疗效果能持续多久 重复服药是必要的。这些实际问题的答案具有巨大的临床后果 有效的CF基因治疗的发展。这一目标将解决AAV1载体包含的程度 AAV1-CBΔ27-264或ΔR-cftr在抢救CF表型中是否有效以及抢救时间长短 在单剂AAV之后会持续下去。目的3：确定使用AAV1载体给药是否会对 截短的cftr将导致雪貂胃肠道和人类肠状突的转导。考虑到 对于CF的基因治疗将必须针对多个器官，这里的目标是证明AAV1衍生的载体将 转导胃肠道。为此，我们将采取双管齐下的方法：首先，从输入的雪貂 目标1和2，我们将分离胃肠道的部分(回肠和结肠)，并测量 被转换的切除的组织。其次，鉴于我们研究的最终目标是治疗CF患者， 我们将研究含有截短cftr的AAV1病毒在多大程度上能从患者手中拯救人类肠道。 这种突变很难用目前的治疗方法来治疗。 意义：慢性萎缩性胃炎是一种常染色体疾病，可导致显著的发病率和死亡率。 精神障碍(9)。这项工作是新颖的，因为它建议治疗与CF相关的多器官病理。

AAV vectors have great potential as gene therapeutic agents (3-5), in particular for cystic fibrosis (CF). Studies originating from our group led to the first use of rAAV in humans (2). Many studies have shown that AAV vectors can be used safely (5,6,9). The major challenge, however, is that they have not achieved a reproducible thera- peutic effect, making it necessary to take a new approach to AAV gene therapy. In previous work, we have identified three new strategies to alleviate these problems: 1) the use of AAV1, which is more tropic for the lung; 2) the use of 27-264, a truncated version of CFTR that corrects ∆F508 by a novel mechanism; and 3) the inclu- sion of a powerful chicken β-actin (CBA) promoter. Given that CF is a multi-organ disease, a gene-based therapy will have to target several organs whose cells normally turn over (3,4), making it advantageous to employ a virus that can transduce multiple cell types if it is to be used in a single-treatment therapy. To further explore this approach, we will use a ferret model bearing the G551D mutation, which responds to the CF potentiator VX-770. We propose three overall Specific Aims: Aim 1: To evaluate systemic vs. airway delivery of AAV1 gene therapy to transduce organs affected by CF. For gene therapy to be effective for CF, it must correct defective CFTR function in several organs in the body besides the airways. The question addressed here is whether transduction can occur in peripheral organs following airway delivery of naturally occurring AAV1. Aim 2: To determine the duration of the therapeutic effects of AAV1 vectors containing truncated CFTR. The overarching questions for CF gene therapy are whether gene transduction can rescue the CF phenotype and how long the therapeutic effect will last before a repeated dose is necessary. The answers to these practical questions have enormous clinical consequences for the development of effective CF gene therapy. This Aim will address the extent to which AAV1 vectors containing either AAV1-CBΔ27-264 or ΔR-CFTR will be effective in rescuing the CF phenotype and how long the rescue will last following a single dose of AAV. Aim 3: To determine whether dosing with an AAV1 vector con- taining a truncated CFTR will lead to transduction in the ferret GI tract and human enteroids. Given that gene therapy for CF will have to target multiple organs, the goal here is to show that AAV1-derived vectors will transduce the GI tract. For this Aim, we will take a two-pronged approach: First, from the ferrets transduced in Aims 1 and 2, we will isolate sections of GI tract (ileum and colon) and measure the transport characteristics of the transduced, excised tissue. Second, given that the ultimate goal of our studies is to treat patients with CF, we will study to what extent AAV1 virus containing truncated CFTR will rescue human enteroids from patients with mutations that are hard to treat with current therapies. Significance: CF is an autosomal disease that leads to significant morbidity and mortality in patients with the disorder (9). This work is novel because it proposes to treat the multiorgan pathologies associated with CF.