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）的医学管理取得了最新进展，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PAH）中产生持久的疗效方面前景广阔。然而，目前的基因递送技术必须经过改造，以提高递送效率，以特异性靶向受影响的器官、组织区室和细胞类型，如PAH中的肺血管系统和血管平滑肌细胞。腺相关病毒（AAV）载体（与腺病毒载体不同）最近获得了显著的关注，因为与大多数载体不同，AAV不引起明显的炎症或毒性反应，同时提供体内长期基因表达。尽管有证据表明重组AAV（rAAV）载体有希望在肺中安全、长期地表达转基因，但尚未确定优化肺血管递送的策略，因此肺血管靶向的rAAV载体不可用。我们在该提议中的目的是鉴定在血管施用后最能使肺脉管系统稳定的AAV血清型（目前使用的AAV血清型，AAV 1 - 9），然后使用定向进化方法进一步修饰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，工程AAV血清型使用定向进化的方法，纳入肺内皮细胞培养中的选择和扩增，将产生新的AAV衣壳变体与增强的向性肺血管。我们将在以下目标中讨论这一假设：具体目标1。确定目前已知的对肺血管内皮细胞具有最大嗜性的AAV血清型（AAV 1 -9）。具体目标2。确定在细胞培养中引入选择和扩增以及体内选择的定向进化是否将产生当通过脉管系统施用时具有增强的肺内皮细胞向性的新型AAV变体。这些特定目标的完成将导致肺内皮细胞特异性载体的开发。肺血管靶向将使得能够开发和实施基因治疗方式作为治疗肺血管疾病（包括但不限于肺动脉高压）的手段。该技术可以推广到产生具有治疗许多疾病的其他器官和/或细胞类型特异性向性的AAV载体。尽管肺动脉高压（PAH）的医疗管理取得了最新进展，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%。腺相关病毒载体最近得到了极大的关注.尽管有证据表明，这些载体有希望在肺部安全，长期的基因治疗使用，战略需要开发，以推动这一领域的发展。这项研究的完成将导致适合于基因治疗的肺病毒载体的开发。此外，这项技术可以推广到开发适合治疗各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.