AAV capsid engineering for enhancing gene transfer

用于增强基因转移的 AAV 衣壳工程

• 批准号: 10574568
• 负责人: Mark A Kay
• 金额: $ 69.68万
• 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/10574568
• 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; Laboratories; Learning; Left; Libraries; Liver; Mammals; Measures; Mediating; Modeling; Molecular; Mus; Mutagens; 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; blood-brain barrier penetration; 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; knockout gene; mouse model; neutralizing antibody; new technology; nonhuman primate; novel; screening; stem; stem cell genes; success; therapeutic DNA; transduction efficiency; 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载体在临床试验中显示出巨大的前景。这些向量代表一个基因 转移/基因组编辑平台不仅有可能治疗遗传疾病，而且有可能治疗多种疾病 获得性疾病，包括感染和感染预防、神经变性以及由此引起的疾病 来自免疫系统功能障碍。转化所取得成功的主要限制步骤之一 人类疾病动物模型与人类载体转导之间缺乏强相关性 物种之间的特性。因为转导很大程度上是由衣壳蛋白的变异决定的 序列，为了获得在人类中具有增强转导特性的衣壳，我们追求多种 物种衣壳改组，以及体外和体内进化选择范例，以创建和识别新的 具有临床相关资产的嵌合衣壳。在本轮融资期间，我们发现了一些 灵长类动物肝脏转导谱增加 10 倍的嵌合衣壳。这些衣壳之一在临床上 试验中，另外两种最近的分离株正在由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，拟议工作的总体目标是建立在 我们努力开发用于新衣壳工程方法的高通量技术，并优化 选择方案。我们的具体目标是创建和鉴定具有增强功能的衣壳：(1) 人类肝脏 转导，（2）穿透人体血脑屏障并转导神经元和 (3)转导人类造血干细胞以提高基因组编辑效率。 我们还将研究用我们的几个新的特定物种观察到的物种选择性背后的机制 嵌合衣壳衍生的载体。在各个屏幕中获得的向量将被进一步 在适当的人源化动物模型或非人类灵长类动物中进行评估。了解到的信息 将有助于我们优化 AAV 介导的人类基因转移的知识。新的衣壳 将可用于临床基因转移/基因组编辑试验。