Pulmonary Vascular Targeting of Gene Delivery Using Directed Evolution of AAV

描述（由申请人提供）：尽管肺动脉高压（PAH）的医疗管理最近取得了进展，但在美国，诊断后1年、2年和3年的生存率分别为80%、76%和49%。目前，唯一的治疗方法是肺移植。另外，基因治疗在肺血管疾病（包括多环芳烃）中产生持久的、可治愈的治疗效果有很大的希望。然而，目前的基因传递技术必须经过改造以提高传递效率，以特异性地靶向受影响的器官、组织腔室和细胞类型，如肺动脉高压中的肺血管和血管平滑肌细胞。腺相关病毒（AAV）载体（不同于腺病毒载体）最近得到了极大的关注，因为与大多数载体不同，AAV不会引起明显的炎症或毒性反应，同时在体内提供长期的基因表达。尽管有证据表明重组AAV （rAAV）载体有望在肺中安全、长期地进行转基因表达，但优化肺血管递送的策略尚未确定，因此肺血管靶向rAAV载体尚不可用。我们的目标是确定在血管给药后最能转导肺血管的AAV血清型（在目前使用的AAV血清型中，AAV1- 9），然后使用定向进化方法进一步修改该血清型以增强肺向性。因此，我们将产生特异性靶向肺血管的新型AAV血清型。我们假设，利用定向进化方法，结合肺内皮细胞培养中的选择和扩增，设计AAV血清型，将产生新的AAV衣壳变体，其对肺血管的趋向性增强。我们将在以下目标中解决这一假设：确定目前已知的AAV血清型（AAV1-9）对肺血管内皮细胞的最大倾向性。具体目标2。确定定向进化结合细胞培养和体内选择和扩增是否会产生新的AAV变体，通过血管给药增强肺内皮细胞的亲和性。完成这些特定目标将导致肺内皮细胞特异性载体的发展。肺血管靶向将使基因治疗模式的发展和实施成为治疗肺血管疾病的一种手段，包括但不限于肺动脉高压。这项技术可以推广到生产具有其他器官和/或细胞类型特异性的AAV载体，用于治疗许多疾病。尽管肺动脉高压（PAH）的医疗管理最近取得了进展，但在美国，诊断后1年、2年和3年的生存率分别为80%、76%和49%。腺相关病毒载体近年来引起了广泛的关注。尽管有证据表明，这些载体有望在肺部进行安全、长期的基因治疗，但需要制定策略来推动这一领域的发展。这项研究的完成将导致适合基因治疗的肺病毒载体的发展。此外，这项技术可以推广到开发新的病毒载体，适用于治疗各种疾病。

DESCRIPTION (provided by applicant): Despite recent advances in the medical management of pulmonary arterial hypertension (PAH), survival rates in the U.S. at one-, two-, and three-years following diagnosis are 80%, 76%, and 49%, respectively. At present, the only curative therapy is lung transplantation. Alternatively, gene therapy has great promise for producing long-lasting, curative therapeutic effects in pulmonary vascular diseases, including PAH. However, current gene delivery technology must be engineered to improve delivery efficiency to specifically target affected organs, tissue compartments, and cell types, such as the pulmonary vasculature and vascular smooth muscle cells in PAH. Adeno-associated viral (AAV) vectors (distinct from adenoviral vectors) have recently gained significant attention, since unlike most vectors, AAV does not elicit appreciable inflammatory or toxic responses, while providing long-term gene expression in vivo. Despite evidence that recombinant AAV (rAAV) vectors have promise for safe, long-term transgene expression in the lung, a strategy to optimize pulmonary vascular delivery has not been defined, and thus pulmonary vascular-targeted rAAV vectors are not available. Our objectives in this proposal are to identify the AAV serotype (of the currently utilized AAV serotypes, AAV1- 9) best able to transduce the lung vasculature following vascular administration and then to further modify this serotype to enhance lung tropism using a directed evolution approach. We will thereby generate novel AAV serotypes that specifically target the pulmonary vasculature. We hypothesize that engineering AAV serotypes using a directed evolution approach, incorporating selection and amplification in pulmonary endothelial cell culture, will yield novel AAV capsid variants with enhanced tropism to the lung vasculature. We will address this hypothesis in the following aims: Specific Aim 1. To determine the currently known AAV serotype (AAV1-9) with the greatest tropism to pulmonary vascular endothelial cells. Specific Aim 2. To determine whether directed evolution incorporating selection and amplification in cell culture and selection in vivo will yield novel AAV variants with enhanced pulmonary endothelial cell tropism when administered via the vasculature. Completion of these specific aims will result in the development of pulmonary endothelial cell specific vectors. Pulmonary vascular targeting will enable the development and implementation of gene therapeutic modalities as a means of treating pulmonary vascular diseases, including but not limited to pulmonary arterial hypertension. This technology can be generalized to produce AAV vectors with other organ and/or cell type specific tropisms that treat many diseases. Despite recent advances in the medical management of pulmonary arterial hypertension (PAH), survival rates in the U.S. at one-, two-, and three-years following diagnosis are 80%, 76%, and 49%, respectively. Adeno- associated viral vectors have recently gained significant attention. Despite evidence that these vectors have promise for safe, long-term gene therapy use in the lung, strategies need to be developed to move this field forward. Completion of this study will result in the development of lung viral vectors suitable for gene therapy. Furthermore, this technology can be generalized to develop new viral vectors suitable to treat a wide-array of diseases.