AAV capsid engineering for enhancing gene transfer

用于增强基因转移的 AAV 衣壳工程

• 批准号: 10352396
• 负责人: Mark A Kay
• 金额: $ 69.74万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10352396
• 关键词: 3-Dimensional; Address; Amino Acid Sequence; Animal Model; Animals; Astrocytes; Back; Behavior; Blood - brain barrier anatomy; Capsid; Capsid Proteins; Cells; Chromatin; Clinic; Clinical Trials; Complex; Cultured Cells; DNA; DNA Sequence; Disease; Engineering; Factor IX; Family suidae; Funding; Gene Transfer; Genetic Diseases; Genome; Genome engineering; Goals; Hematopoietic stem cells; Hemophilia B; Hepatocyte; Human; Humoral Immunities; Immune System Diseases; In Vitro; Infection; Integration Host Factors; Knock-out; Laboratories; Left; Libraries; Liver; Mammals; Measures; Mediating; Modeling; Molecular; Mus; Nerve Degeneration; Neurons; Organoids; Penetration; Peptides; Plasma; Preclinical Testing; Primates; Property; Prophylactic treatment; Recombinant adeno-associated virus (rAAV); Recombinants; Rodent; Scheme; Serum; System; Techniques; Technology; Testing; Time; Transgenic Organisms; Translating; Variant; Viral; Viral Genome; Virus; Work; adeno-associated viral vector; cell type; clinically relevant; design; embryonic stem cell; gene therapy; genome editing; high throughput screening; high throughput technology; human disease; human model; human tissue; humanized mouse; improved; in vivo; induced pluripotent stem cell; mouse model; neutralizing antibody; new technology; nonhuman primate; novel; screening; stem; stem cell genes; success; therapeutic DNA; vector

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.

重组AAV矢量在临床试验中表现出了很大的希望。这些向量代表一个基因 转移/基因组编辑平台，不仅可以治疗遗传疾病，而且有可能治疗无数的遗传疾病 获得的疾病，包括感染和预防感染，神经退行性的疾病以及导致的疾病 免疫系统功能障碍。翻译成功取得成功的主要限制步骤之一 人类疾病的动物模型是载体转导之间缺乏强大的相关性 物种之间的特性。因为转导大部分取决于衣壳蛋白的变化 序列，为了获得具有增强的人类透性特性的衣壳，我们追求了多种 物种带子式洗牌，体外和体内进化选择范例，以创建和识别新颖 带有临床相关资产的嵌合带壳。在当前的资金期间，我们发现了几个 灵长类动物的肝脏转导谱增加了10倍的嵌合囊膜。这些衣壳之一是临床 试验和最近的两个分离株正在商业和学术中心进行晚期临床前测试。然而 即使这些改进的AAV矢量似乎也没有达到可以实现的相同水平的转导水平 在具有其他已建立的AAV衣壳的啮齿动物中。因此，拟议工作的一般目标是建立 我们为开发新的Capsid工程方法开发高通量技术的努力，并优化 选择方案。我们的具体目标是创建和识别增强的衣壳：（1）人类肝脏 转导，（2）通过人脑屏障的穿透以及神经元的转导和转导 星形胶质细胞和（3）人类造血干细胞的转导，以提高基因组编辑效率。 我们还将研究与我们的几个新特定观察到的物种选择性背后的机制 嵌合带胶囊衍生的载体。在各个屏幕中获得的向量将进一步 在适当的人道化动物模型或非人类灵长类动物中进行评估。所学的信息 将有助于我们的知识，以优化人类中的AAV介导的基因转移。新的衣壳 将可用于临床基因转移/基因组编辑试验。