Selection of New rAAV Vectors Using Replicating Viral Capsids Libraries

 产品说明：重组腺相关病毒载体（rAAV）在血友病B、遗传性失明和脂蛋白脂酶缺乏症等疾病中显示出良好的临床效果。即使在治疗“简单”疾病实体的早期有限成功， 是电流矢量的一些限制。例如，在血友病B试验中，在IV输注后，AAV 8-hFIX转导患者肝脏，并且证明出血素质的改善，但是仍然存在以下困难：1）一些患者发生短暂的转氨酶升高，认为是由于针对AAV 8衣壳的T细胞应答; 2）基于载体剂量/体重的转基因表达远低于小鼠研究（~ 10倍）和非人灵长类动物研究（~3- 5倍）的预测，表明人肝细胞中的转导不是最佳的; 3）在约1/3的患者中发现预先存在的中和性抗AAV 8抗体; 4）可能需要重新施用不同的载体; 5）AAV基因组包装大小的限制使得难以或甚至不可能治疗其他类似疾病（例如血友病A，血友病的更常见形式）。由于rAAV转导中的显著差异由衣壳氨基酸序列中的小变化决定的事实，我们已经进行了衣壳基因的分子改组以创建具有广泛序列变体（~ 10 e7）的AAV文库。这些新型衣壳取代病毒质粒中的野生型衣壳，并用于在辅助病毒存在下产生感染性病毒。复制的AAV在选择性条件下生长，我们根据所需的结果而改变，并且分离具有选择性优势的那些并确定它们的序列。然后，这些衣壳可用于包装治疗基因并确定其转导。我们和其他人已经选择了具有新特性的AAV衣壳，其中许多对基因治疗界非常有用。然而，我们认为复制衣壳文库的全部效用尚未达到。我们计划组装新的更复杂的文库，并在不同选择压力下在嵌合小鼠-人肝模型中维持的原代人肝细胞中连续传代后选择富集的衣壳。我们将从大多数选择的衣壳载体和研究他们的转导在体外和体内。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）选择具有新颖细胞特异性的衣壳（例如仅转导被丙型肝炎病毒感染的肝细胞的AAV）; 2）选择可包装较大基因组的衣壳，从而扩大AAV载体用于其表达盒刚好超过当前包装限制的疾病的效用（例如囊性纤维化、杜氏肌营养不良和血友病A）。我们相信这项工作不仅将提供新的AAV载体，用于研究和临床基因治疗的扩大使用，而且还将提供重要的定量指导方针，使AAV衣壳改组更有效地为所有从事这一研究的群体。

 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.