AAV capsid engineering for enhancing gene transfer

重组AAV载体在临床试验中显示出巨大的前景。这些载体代表一个基因 转移/基因组编辑平台，不仅有可能治疗遗传疾病， 获得性疾病，包括感染和感染预防，神经变性，以及由此导致的疾病， 免疫系统功能紊乱将2004年取得的成功转化为2005年取得的成功的主要限速步骤之一是， 人类疾病的动物模型对人类缺乏强相关性的载体转导 物种之间的属性因为转导在很大程度上取决于衣壳蛋白的变异 序列，为了获得在人类中具有增强的转导特性的衣壳，我们已经进行了多- 物种衣壳改组，以及体外和体内进化选择范例，以创建和鉴定新的 具有临床相关资产的嵌合衣壳。在目前的融资期间，我们发现了几个 嵌合衣壳具有10倍增加的灵长类动物肝脏转导谱。其中一个衣壳在临床上 最近的两个分离株正在商业和学术中心进行后期临床前测试。然而 即使这些改进的AAV载体似乎也没有达到可以实现的相同水平的转导 在啮齿动物中与其他已建立的AAV衣壳。因此，拟议工作的总目标是在 我们努力开发高通量技术，用于新的衣壳工程方法，并优化 选择方案。我们的具体目标是创造和鉴定衣壳，增强：（1）人类肝脏 转导，（2）穿透人血脑屏障和神经元的转导， 星形胶质细胞，和（3）转导人造血干细胞以提高基因组编辑效率。 我们还将研究我们的几个新的特定的物种选择性背后的机制， 嵌合衣壳衍生载体。在各个屏幕中获得的矢量将进一步 在适当的人源化动物模型或非人灵长类动物中评价。了解到的信息 将有助于我们对优化人类AAV介导的基因转移的知识。新的衣壳 将用于临床基因转移/基因组编辑试验。

Recombinant AAV vectors have shown great promise in clinical trials. These vectors represent a gene transfer/genome editing platform that has the potential to treat not only genetic diseases but a myriad of acquired disorders that include infection and infection prophylaxis, neurodegeneration, and diseases resulting from immune system dysfunction. One of the major rate-limiting steps in translating the success achieved in animal models of human disease to humans is the lack of a strong correlation between vector transduction properties between species. Because transduction is dictated in large part by variations in the capsid protein sequence, in order to obtain capsids with enhanced transducing properties in humans we have pursued multi- species capsid shuffling, and in vitro and in vivo evolutionary selection paradigms to create and identify novel chimeric capsids with clinically relevant assets. During the current funding period, we discovered several chimeric capsids with a 10-fold increased primate liver transduction profile. One of these capsids is in clinical trials, and two more recent isolates are in late preclinical testing by commercial and academic centers. Yet even these improved AAV vectors do not appear to reach the same level of transduction that can be achieved in rodents with other established AAV capsids. Thus, the general goal of the proposed work is to build upon our efforts to develop high throughput technologies for new capsid engineering approaches, and optimized selection schemes. Our specific goals are to create and identify capsids that have enhanced: (1) human liver transduction, (2) penetration through the human blood brain barrier and transduction of neurons and astrocytes, and (3)transduction of human hematopoietic stem cells for increased genome editing efficiencies. We will also study the mechanism behind the species selectivity observed with several of our new specific chimeric capsid derived vectors. The vectors that are obtained in the respective screens will be further evaluated in either an appropriate humanized animal model or non-human primates. The information learned will contribute to our knowledge towards optimizing AAV-mediated gene transfer in humans. The new capsids will be made available for use in clinical gene transfer/genome editing trials.