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

AAV载体具有作为基因治疗剂的巨大潜力（3-5），特别是对于囊性纤维化（CF）。研究 源自我们小组的研究导致rAAV首次用于人类（2）。许多研究表明，AAV载体 可以安全使用（5，6，9）。然而，最大的挑战是，他们还没有实现可再生的治疗， 因此，有必要对AAV基因治疗进行新的探索。在以前的工作中，我们有 确定了三种新的策略来缓解这些问题：1）使用对肺更有向性的AAV 1; 2)27-264的使用，CFTR的截短版本，其通过新的机制校正CFNF 508;和3）包括- 强有力的鸡β-肌动蛋白（CBA）启动锡永。鉴于CF是一种多器官疾病， 将必须靶向细胞正常翻转的几个器官（3，4），这使得使用病毒是有利的 如果它被用于单一治疗，它可以复制多种细胞类型。为了进一步探讨这一点， 方法，我们将使用携带G551 D突变的雪貂模型，其响应于CF增效剂VX-770。 我们提出了三个总体具体目标： 目的1：评价AAV 1基因治疗对受感染器官的全身与气道递送， 参见为了使基因治疗对CF有效，它必须纠正CF中几个器官的CFTR功能缺陷。 除了呼吸道。这里讨论的问题是转导是否可以发生在外周器官 在天然存在的AAV 1的气道递送之后。目的2：确定治疗的持续时间 含有截短的CFTR的AAV 1载体的作用。CF基因治疗的首要问题是 基因转导是否能挽救CF表型，治疗效果能持续多久， 重复剂量是必要的。这些实际问题的答案有巨大的临床后果， 开发有效的CF基因治疗。该目的将解决含有AAV 1载体的AAV 1载体在多大程度上可用于治疗癌症的问题。 AAV 1-CBΔ27-264或ΔR-CFTR将有效挽救CF表型，并且挽救需要多长时间 将在单剂量AAV后持续。目的3：为了确定给药AAV 1载体是否能与 获得截短的CFTR将导致在雪貂胃肠道和人肠样组织中的转导。鉴于 CF的基因治疗必须靶向多个器官，这里的目标是表明AAV 1衍生的载体将 胃肠道扩张。为此，我们将采取双管齐下的方法：首先，从转导的雪貂中， 目的1和2，我们将分离胃肠道（回肠和结肠）的部分，并测量 被转换的切除组织其次，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AAV 1病毒将在多大程度上从患者中拯救人类肠上皮细胞 这些突变很难用目前的疗法治疗。 意义：CF是一种常染色体疾病，在患有CF的患者中导致显著的发病率和死亡率。 无序（9）。这项工作是新颖的，因为它提出了治疗与CF相关的多器官病变。

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.