Engineering improved delivery and immune profiles of Cas9 orthologues for gene therapy

工程改进了用于基因治疗的 Cas9 直向同源物的递送和免疫特征

• 批准号: 10444901
• 负责人: Elizabeth Stahl
• 金额: $ 5.83万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10444901
• 关键词: Address; Affect; Age-Months; Amyotrophic Lateral Sclerosis; Animal Model; Base Sequence; Bilateral; Biological Assay; Biotechnology; Blood; Blood - brain barrier anatomy; Brain; CRISPR/Cas technology; Capsid; Cations; Cells; Cellular Immunity; Cessation of life; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Corpus striatum structure; DNA; DNA Double Strand Break; DNA delivery; Dependovirus; Disease; Dose; Engineering; Enzyme Kinetics; Enzyme-Linked Immunosorbent Assay; Enzymes; Equilibrium; Event; Exclusion; Exhibits; Flow Cytometry; Genes; Genetic Diseases; Genetic Materials; Genetic Recombination; Genome; Goals; Gold; Guide RNA; Human; Humoral Immunities; Immune; Immune response; Immunity; In Vitro; Injections; Kinetics; Laboratories; Liver; Location; Long-Term Effects; Mass Behavior; Measures; Medicine; Methods; Molecular Biology; Motor; Mus; Mutation; Neuraxis; Neurons; Neurosciences; Nuclear; Occupations; Organ; Outcome; Patients; Peripheral; Protein Engineering; Proteins; Publications; Publishing; Reporter; Research Project Grants; Research Proposals; Retina; Ribonucleoproteins; Risk; Safety; Serum; Site; Staphylococcus aureus; Streptococcus pyogenes; System; Technical Expertise; Testing; Therapeutic; Therapeutic Uses; Time; Training; Translating; Tropism; Variant; Virus Integration; Visualization; Work; base; cost; delivery vehicle; endonuclease; experimental study; gene correction; gene therapy; genome editing; genotoxicity; human subject; immunogenicity; improved; in vivo; in vivo Model; interest; mouse model; nerve stem cell; nuclease; premature; promoter; screening; side effect; superoxide dismutase 1; tool; transgene delivery; transgene expression; viral DNA

Project Summary / Abstract The current gold standard to deliver gene therapies, adeno-associated virus (AAV), has shown safe and stable transgene expression in many applications. However, the combination of AAV with CRISPR introduces unique risks of genotoxic side effects from long-term nuclease expression and integration of viral DNA into sites of DNA breaks in the host genome. Furthermore, the ssDNA genome of AAV limits its packaging capacity so that two AAVs must be used to deliver Cas9 from Streptococcus pyogenes (SpyCas9) and its guide RNA, limiting efficiency and increasing costs. Interest has switched to using a smaller Cas9 orthologue derived from Staphylococcus aureus (SauCas9), although several enzyme features have been shown to be significantly different. Furthermore, pre-existing immunity to AAV capsids, as well as SpyCas9 and SauCas9, has been identified in humans, potentially limiting the therapeutic use of these molecules. While the host response to the two orthologues is dependent on previous exposure, SauCas9 was found to elicit a stronger immune response than SpyCas9 in human subjects when measured by immunoblot, ELISA, and ELISpot assays. Therefore, although SauCas9 is a smaller nuclease that enables delivery by AAV, there are several questions about safety that must be addressed. Previous work in the Doudna laboratory has shown that the SpyCas9 endonuclease can be engineered with cationic residues to make the ribonucleoprotein (RNP) inherently cell-penetrating in neural precursor cells in vitro and in neurons in vivo by non-viral delivery. I have now engineered SauCas9 to also act as a cell-penetrating RNP. The purpose of this research proposal is to evaluate the outcomes of engineered Cas9 orthologues delivered as RNPs or AAVs in the mammalian brain. Aim 1 employs an unbiased protein engineering and screening strategy to make small deletions across Cas9 that improve the cellular host immune response to the protein. Aim 2 will test these variants as ribonucleoproteins or as adeno-associated viruses delivered by stereotaxic injection into the striatum of a fluorescent reporter animal model to assess genome editing outcomes and the host immune response; and Aim 3 will apply these findings to genetically correcting SOD1 mutations in an animal model of amyotrophic lateral sclerosis. Taken together, developing a cell-penetrating and immune-stealthy Cas9 RNP for transient and local genome editing would improve the safety of CRISPR therapies and accelerate the pace of clinical trials that could immediately benefit patients.

项目摘要 /摘要 提供基因疗法的当前黄金标准，腺相关病毒（AAV）已显示出安全稳定 在许多应用中的转基因表达。但是，AAV与CRISPR的组合引入了独特 长期核酸酶表达和病毒DNA整合到DNA位点的遗传毒性副作用的风险 宿主基因组中断。此外，AAV的SSDNA基因组限制了其包装能力，因此两个 必须使用AAV从链球菌为链球菌（spycas9）及其引导RNA传递Cas9，限制 效率和成本提高。兴趣已转换为使用较小的Cas9直系同源器 金黄色葡萄球菌（SAUCAS9），尽管已经证明了几种酶特征显着 不同的。此外，对AAV Capsids以及Spycas9和Saucas9的免疫力已经存在 在人类中发现，可能限制了这些分子的治疗使用。主机对 两个直系同源物取决于先前的暴露，发现saucas9引起了更强的免疫反应 当通过免疫印迹，ELISA和ELISPOT测定法测量时，人类受试者中的spycas9。所以， 尽管saucas9是一个较小的核酸酶，可以通过AAV传递，但有关安全的问题有几个问题 那必须解决。 Doudna实验室的先前工作表明SpyCAS9核酸内切酶 可以用阳离子残留物进行设计，以使核糖核蛋白（RNP）固有地在细胞中渗透 通过非病毒递送在体外和体内神经元中的神经前体细胞。我现在已经设计了saucas9 还充当细胞渗透RNP。这项研究建议的目的是评估 哺乳动物大脑中以RNP或AAV为单位的工程CAS9直系同源物。 AIM 1采用公正 蛋白质工程和筛选策略，使CAS9跨CAS9进行小删除，以改善细胞宿主 对蛋白质的免疫反应。 AIM 2将测试这些变体作为核糖核蛋白或腺相关 立体定位注射输送到荧光报告动物模型的纹状体中输送的病毒以评估 基因组编辑结果和宿主免疫反应； AIM 3将把这些发现应用于基因 纠正肌萎缩性侧硬化症动物模型中的SOD1突变。两者一起开发 用于瞬时和局部基因组编辑的细胞渗透和免疫侵蚀性CAS9 RNP将提高安全性 CRISPR疗法并加速了可以立即使患者受益的临床试验的速度。