AAV Core

载体核心的主要目标是提供高质量的自互补(Sc)和单链(Ss)rAAV1载体，并从血清学上筛选适合rAAV1抗病毒研究的NHP动物。我们将通过以下具体目标实现这些目标： 目的1.设计、制作、生产和质控不同规模的单链AAV1载体批次，包括多种转基因和表达盒，以满足本项目其他研究人员的特殊需求。更具体地说，120只恒河猴将在5年内参加不同的研究，数百只小鼠将用于猕猴前评估。平均而言，我们估计每年将生产15-20个载体批次，以满足这些研究的需求。 目的2.通过体外和体内中和抗体(NAB)试验，筛选NHP人群对AAV1的预先免疫，以选择AAV1-NAB阴性动物进行疫苗和治疗研究。对NHP群体进行预筛选，以选择不存在rAAV1中和抗体(NAB)的动物，是rAAV1介导的抗HIV免疫粘附素基因转移的关键。我们的数据表明，NHP人群中灵长类AAVs的血清学患病率在60%-80%之间。这意味着我们可能需要对360多只动物进行筛查，才能确定120只动物没有AAV1NAB。 目的3.为更大规模的NHP翻译研究和未来基于rAAV1的抗HIV疫苗和治疗药物的临床开发提供新的、可扩展的rAAV生产方法。我们目前的AAV生产系统应该可以满足该计划项目早期的媒介需求。然而，大规模的载体生产可能成为更大的翻译NHP研究和未来临床开发的瓶颈。我们将利用我们在开发各种载体包装细胞系和基于感染的载体生产系统方面的丰富经验，开发一种基于293细胞感染的新颖且可扩展的生产方法来克服这一限制。

The main objectives of the Vector Core are to provide high quality self-complementary (sc) and single-stranded (ss) rAAV1 vectors and serologically screen NHP animals suitable for rAAV1 anti-viral studies. We will accomplish these goals through the following specific aims: Aim 1. To design, create, produce and quality control test scAAV1 vector lots at different scales with a variety of transgenes and expression cassettes to serve the specific needs of other investigators of this program project. More specifically, 120 rhesus macaques will be enrolled for different studies over 5 years and hundreds of mice will used for pre-macaque evaluation. In average, we estimate that 15-20 vector lots will be produced annually to meet the needs of those studies. Aim 2. To screen NHP populations for pre-existing immunity against AAV1 by using both in vitro and in vivo neutralizing antibody (NAB) assays to select AAV1-NAB free animals for vaccine and therapeutic studies. Pre-screening of NHP population to select the animals without preexisting neutralizing antibody (NAB) to rAAV1 is essential for rAAV1-mediated anti-HIV immunoadhesin gene transfer. Our data suggested that the serological prevalence of primate-derived AAVs in NHP populations ranges from 60-80%. This implies that we may have to screen more than 360 animals to identify 120 animals free of AAV1 NAB. Aim 3. To develop novel and scalable rAAV production method for larger scale translational NHP studies and future clinical development of rAAV1-based anti- HIV vaccine and therapeutics. Our current AAV production system should meet the vector needs in the early stage of this program project. However, large scale vector production may become a bottle neck for larger translational NHP studies and future clinical development as well. We will utilize our extensive experience in developing various vector packaging cell lines and infection-based vector production system to develop a 293 cell infection-based novel and scalable production method to overcome this limitation.