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.

项目总结/摘要 目前基因治疗的金标准，腺相关病毒（AAV），已经显示出安全和稳定 转基因表达在许多应用中。然而，AAV与CRISPR的组合引入了独特的 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风险 宿主基因组的断裂此外，AAV的ssDNA基因组限制了其包装能力，使得两个 AAV必须用于递送来自化脓性链球菌（SpyCas 9）的Cas9及其指导RNA，限制了 效率和增加成本。兴趣已经转向使用来自以下的较小的Cas9直系同源物： 金黄色葡萄球菌（SauCas 9），尽管已经显示出几种酶特征显著地抑制了该酶的活性。 不同.此外，已经证实了对AAV衣壳以及SpyCas 9和SauCas 9的预先存在的免疫性。 在人类中鉴定，潜在地限制了这些分子的治疗用途。虽然主机响应 两种直系同源物依赖于先前的暴露，发现SauCas 9引起更强的免疫应答， 当通过免疫印迹、ELISA和ELISpot测定法测量时，在人类受试者中，因此，我们认为， 尽管SauCas 9是一种能够通过AAV递送的较小核酸酶，但存在几个关于安全性的问题， 必须加以解决。Doudna实验室先前的工作表明，SpyCas 9内切核酸酶 可以用阳离子残基进行工程改造，使核糖核蛋白（RNP）固有地穿透细胞， 体外神经前体细胞和通过非病毒递送体内神经元。我现在设计了SauCas 9 也作为细胞穿透RNP。本研究提案的目的是评估 工程化的Cas9直系同源物在哺乳动物大脑中以RNP或AV形式递送。目标1采用无偏 蛋白质工程和筛选策略，以在Cas9上进行小的缺失，从而改善细胞宿主 对蛋白质的免疫反应。目的2将测试这些变异作为核糖核蛋白或作为腺相关 通过立体定位注射将病毒递送到荧光报告动物模型的纹状体中，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遗传学 在肌萎缩侧索硬化症的动物模型中校正SOD 1突变。综合考虑， 用于瞬时和局部基因组编辑的细胞穿透和免疫隐形Cas9 RNP将提高安全性， CRISPR疗法，并加快临床试验的步伐，使患者立即受益。