Selection of New rAAV Vectors Using Replicating Viral Capsids Libraries

 描述：重组腺相关病毒载体(RAAV)在治疗血友病B、遗传性失明和脂蛋白脂肪酶缺乏症等疾病方面显示出良好的临床效果。即使早期在治疗“简单”疾病方面取得了有限的成功， 是当前向量的一些限制。例如，在血友病B试验中，AAV8-hFIX在静脉输注后转导患者肝脏，出血质量得到改善，但仍然存在以下困难：1)一些患者发展为一过性转氨炎，被认为是由于针对AAV8衣壳的T细胞反应；2)基于载体每体质量的转基因表达远低于小鼠研究(~10倍)和非人类灵长类动物研究(~3-5倍)，表明在人类肝细胞中转导并不理想；3)约1/3的患者发现预先存在的中和抗AAV8抗体；4)可能需要重新注射不同的载体；5)AAV基因组包装的大小限制使得很难甚至不可能治疗其他类似的疾病(例如，血友病A，更常见的血友病形式)。由于rAAV转导的显著差异是由衣壳氨基酸序列的微小变异决定的，我们对衣壳基因进行了分子改组，以创建具有广泛序列变体(~10e7)的AAV文库。这些新的衣壳取代了病毒质粒中的野生型衣壳，并在辅助病毒存在的情况下用于产生传染性病毒。复制型AAV是在选择性条件下培养的，我们根据期望的结果而变化，那些具有选择性优势的AAV被分离出来并确定它们的序列。这些衣壳然后可以用来包装治疗性基因，并确定它们的转导。我们和其他人选择了具有新特性的AAV衣壳，其中许多对基因治疗社区非常有用。然而，我们觉得复制衣壳文库的全部效用还没有达到。我们计划组装新的更复杂的文库，并在不同选择压力下，在原代人肝细胞中连续传代后，选择不同选择压力下维持的人-鼠嵌合肝细胞中丰富的衣壳。我们将从最精选的衣壳中制作载体，并研究它们在体外和体内的转导。最后，我们将在两个高风险、高回报的筛选中使用这些文库：1)选择具有新的细胞特异性的衣壳蛋白(例如，AAV只转导感染了丙型肝炎病毒的肝细胞)；2)选择可以包装更大基因组的衣壳蛋白，从而扩大AAV载体对那些表达盒刚刚超过当前包装限制的疾病(例如囊性纤维化、杜氏肌营养不良和血友病A)的效用。我们相信，这项工作不仅将为扩大AAV载体在研究和临床基因治疗中的应用提供新的载体，还将为所有从事这一研究的群体提供重要的量化指南，使AAV衣壳洗牌更有效。

 DESCRIPTION: Recombinant adeno-associated viral vectors (rAAV) are showing promising clinical results in a few diseases including hemophilia B, hereditary blindness and lipoprotein lipase deficiency. Even with the early limited success in treating "simple" disease entities, there are a number of limitations with the current vectors. For example, in the hemophilia B trials, AAV8-hFIX transduced patient livers after IV infusion and an improvement in the bleeding diathesis was demonstrated, yet the following difficulties remain: 1) some patients develop a transient transaminitis thought to be due to a T-cell response directed against AAV8 capsids; 2) transgene expression based on vector dose per body mass is far less than predicted from mouse studies (~10x) and non- human primate studies (~3-5x), suggesting transduction in human hepatocytes is not optimal; 3) pre-existing neutralizing anti-AAV8 antibodies are found in about 1/3 of patients; 4) re-administration of a different vector maybe required; 5) a limitation i the size of AAV genome packaging makes it difficult or even impossible to treat other similar diseases (e.g. hemophilia A, the more common form of hemophilia). Due to the fact that significant differences in rAAV transduction are dictated by small variations in capsid amino acid sequences, we have pursued molecular shuffling of capsid genes to create AAV libraries with extensive sequence variants (~10e7). These novel capsids replace the wild-type capsid in a viral plasmid and are used to produce infectious virus in the presence of a helper virus. Replicating AAV is grown under selective conditions that we vary according to the desired outcome, and those with a selective advantage are isolated and their sequences determined. These capsids can then be used to package therapeutic genes and their transduction determined. We and others have selected AAV capsids with new properties, many of which have become very useful to the gene therapy community. However, we feel the full utility of replicating capsid libraries has yet to be reached. We plan to assemble new more complex libraries and select for enriched capsids after serial passage in primary human hepatocytes maintained in a chimeric mouse-human liver model under different selection pressures. We will make vectors from the most selected capsids and study their transduction in vitro and in vivo. Finally, we will use these libraries in two high-risk, high-payoff screens: 1) select capsids with novel cell specificity (e.g. an AAV that only transduces hepatocytes infected with hepatitis- C virus); 2) select capsids that can package larger genomes, thus expanding the utility of AAV vectors for diseases whose expression cassette just exceeds current packaging limits (e.g. cystic fibrosis, Duchenne muscular dystrophy and hemophilia A,). We believe this work will not only provide new AAV vectors for expanded use in research and clinical gene therapy, but will also provide important quantitative guidelines to make AAV capsid shuffling more efficient for all groups pursuing this line of research.