Rational and Combinatorial Engineering of AAV Vectors

描述(由申请人提供)：临床上可用的AAV载体数据强调了在AAV衣壳、基因组和转基因盒水平上继续优化努力的重要性。这项建议的一个重点是获得最适合于系统性疾病(MPS、Hurler等)的临床AAV载体。在衣壳水平上，很明显，动物 模型并不总是预测人类的结果，需要更有效的人类特异性衣壳才能达到较低的给药剂量。在目标1中，我们试图通过在小鼠、灵长类和人类起源的组织上建立第一个AAV受体表达图谱来创建用于人类转导的AAV载体选择的新范式。这一组织特异性AAV受体图谱将被AAV结合和转导数据覆盖，以努力梳理出在不同背景下对组织特异性相互作用重要的衣壳区域。此外，从小鼠模型中建立的灵长类和人类肝脏定向进化策略中分离出的新型嵌合衣壳将与我们的受体/结合图谱进行比较，以确定体外结合是否与体内结果相关。然后，我们将在体内研究灵长类动物肝脏转导的灵长类动物模型中的衣壳分离物，以确定这一策略是否代表了一种有效的方法来获得灵长类动物(人和非人类)肝脏特异性AAV衣壳。在AAV基因组水平上，我们组装了一组依赖DNA修复的AAV底物，这些底物报告了基因组持久性的关键方面，包括环化、连结和同源定向退火。对这些试剂在不同DNA修复途径缺陷的突变背景中的研究将提供对体外和体内对同源重组和非同源末端连接机制的偏好依赖的洞察，提供对疾病环境中载体性能的更好预测(目标2)。在载体转基因的水平上，我们在小鼠的肝脏、心脏和眼睛中展示了一种使用IVS2-654内含子和反义寡核苷酸来诱导转基因合成的新方法。这里的工作旨在产生更小的合成变异体，显示出更严格的控制以及改变的转基因表达水平，从而提供一组可以为特定应用量身定做的调控开关。最后，提出了一种设计“关”开关的策略。 从IVS2-654诱导的转基因合成，这也可能允许在固定的载体剂量下精确调节转基因合成。总之，拟议的实验结果旨在通过我们在衣壳和基因组以及转基因DNA盒水平的持续优化努力，解决在AAV基因治疗应用于系统性疾病方面的临床缺陷。

DESCRIPTION (provided by applicant): The available data of AAV vectors in the clinic emphasize the importance of continued optimization efforts at the levels of the AAV capsid, genome and transgenic cassette. A focus of this proposal is to derive clinical AAV vector best suited for systemic disorders (MPS, Hurlers, etc.). At the capsid level, it is apparent that animal models do not always predict the human outcome and that more efficient human specific capsids are required to achieve a lower administered dose. In Aim 1, we seek to create a new paradigm of AAV vector selection for human transduction by generating the first AAV receptor expression map on tissues of mouse, primate and human origin. This tissue specific AAV receptor Atlas will be overlaid with AAV binding and transduction data in an effort to tease out regions of the capsid important for tissue specific interactions in varied backgrounds. In addition novel chimeric capsids isolated from a directed evolution strategy on primate and human livers established in a mouse model will be triaged against our receptor/binding atlas to determine if in vitro binding correlates to in vivo results. Then, capsid isolates from a primatized-liver mouse model will be investigated for primate liver transduction in vivo to determine if this strategy represents a valid method to derive primate (human & non human) liver specific AAV capsids. At the level of the AAV genome, we have assembled a panel of DNA repair dependent AAV substrates that report critical aspects of genome persistence including circularization, concatemerization and homology directed annealing. Investigations of these reagents in mutant backgrounds defective in different DNA repair pathways will offer insights into the preferred reliance on homologous recombination and non-homologous end joining mechanisms in vitro and in vivo providing a better prediction of vector performance in diseased settings (Aim 2). At the level of the vector transgene, we demonstrate in mouse liver, heart and eye a novel method to induce transgene synthesis using the IVS2- 654 intron and an anti-sense oligonucleotide. The work herein seeks to generate smaller synthetic variants that exhibit tighter control as well as altered transgene expression levels, thus providing a panel of regulatory switches which can be tailored for specific applications. Finally, a strategy is proposed to engineer an "off" switch for the induced transgene synthesis from IVS2-654, which may also allow the precise tuning of transgene synthesis at a fixed vector dose. Collectively, the results of the proposed experiments seek to address the observed clinical deficiencies in AAV gene therapy applications for diseases of systemic nature by our continued optimization efforts at the levels of the capsid and genome as well as the transgenic DNA cassette.