Pulmonary Vascular Targeting of Gene Delivery Using Directed Evolution of AAV

描述(由申请人提供)：尽管最近在肺动脉高压(PAH)的医疗管理方面取得了进展，但在美国，确诊后一年、两年和三年的存活率分别为80%、76%和49%。目前，唯一有效的治疗方法是肺移植。或者，基因治疗在包括PAH在内的肺血管疾病中产生持久的治疗效果具有很大的前景。然而，目前的基因传递技术必须提高传递效率，以专门针对受影响的器官、组织间隔和细胞类型，如PAH中的肺血管和血管平滑肌细胞。腺相关病毒(AAV)载体(有别于腺病毒载体)最近引起了广泛的关注，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，而是在体内提供长期的基因表达。尽管有证据表明，重组AAV(RAAV)载体有望在肺内安全、长期地表达转基因，但优化肺血管递送的策略尚未确定，因此以肺血管为靶点的rAAV载体不可用。我们在这项建议中的目标是确定(目前使用的AAV血清型中的AAV1-9)最能转导血管给药后肺血管的AAV血清型，然后利用定向进化方法进一步修改该血清型以增强肺的趋向性。因此，我们将产生针对肺血管系统的新型AAV血清型。我们假设，使用定向进化方法设计AAV血清型，结合肺内皮细胞培养中的选择和扩增，将产生新的AAV衣壳变异体，其对肺血管的趋向性增强。我们将从以下几个方面阐述这一假说：具体目标1.确定目前已知的AAV1-9对肺血管内皮细胞有最大趋向性的AAV血清型。具体目的2.确定定向进化结合细胞培养中的选择和扩增以及体内选择是否会产生通过血管系统给药时具有增强肺内皮细胞趋向性的新型AAV变异体。这些特定目的的完成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开发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/或细胞类型特异性取向的AAV载体，用于治疗许多疾病。尽管最近在肺动脉高压(PAH)的医疗管理方面取得了进展，但在美国，确诊后一年、两年和三年的存活率分别为80%、76%和49%。腺相关病毒载体最近得到了极大的关注。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，但仍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开发出适合基因治疗的肺病毒载体。此外，这项技术可以推广到开发适合治疗多种疾病的新病毒载体。

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.