Define and minimize the immunogenicity of CRISPR-Cas nucleases

项目摘要 体内CRISPR-Cas基因组编辑具有通过直接纠正 体内受影响细胞的致病突变。CRISPR Cas核酸酶允许位点特异性和 对广泛的生物医学重要细胞靶点进行有效的修饰。葡萄球菌Cas9 金黄色葡萄球菌(S.aureus；SaCas9)是第一个在哺乳动物小细胞中发现活性的同源基因 足以在AAV矢量中进行编码。以前的研究，使用编码在AAV载体中的SaCas9来治疗 幼犬的杜氏肌营养不良症(DMD)导致了这种疾病的功能矫正。 论证了CRISPR可以阻止DMD的进展，恢复功能和 因此，可以成功地用于体内研究。 然而，威胁到有希望的体内基因组编辑治疗性的持久性的一个主要问题 使用Cas9的策略，是表达编辑细胞的Cas9的潜在免疫排斥反应。原有的自适应 对人类常见病原体Cas9变种金黄色葡萄球菌的免疫已有报道。因此，人类细胞 接受SACas9治疗的患者可能会引发适应性记忆免疫反应 细胞毒T细胞对表达Cas9的细胞的杀伤作用。 虽然已经鉴定出SaCas9特异性T细胞，但对SaCas9的获得性免疫反应尚未得到证实 完全有特点的。人类对SaCas9的初级T细胞反应将通过探测人血清进行重新修饰 健康和人类白细胞抗原(人类白细胞抗原)型人献血者(目标1)。此外，要识别小说 和非免疫原性SaCas9变异体，我们将进行全面的突变筛查，以阐明 SaCas9的结构与功能关系。这将为生成通用的SaCas9奠定基础 对多种人类白细胞抗原类型可能无免疫原性(目标2)。 拟议的工作将拓宽基因编辑界的知识，并扩大其影响 为CRISPR/Cas在基因治疗中的临床应用奠定基础。

Project Abstract In vivo CRISPR-Cas genome editing has the potential to transform human medicine by directly correcting disease-causing mutations in affected cells in the body. CRISPR Cas nucleases allow for site-specific and efficient modifications of a wide range of biomedically important cellular targets. Cas9 from Staphylococcus aureus (S.aureus; SaCas9) was the first orthologue discovered to be active in mammalian cells that is small enough to be encoded in an AAV vector. Previous studies, using SaCas9 encoded in AAV vectors to treat Duchenne's Muscular Dystrophy (DMD) in young dogs has resulted in the functional correction of the disease. Demonstrating, the proof-of-concept that CRISPR can halt the progression of DMD and restore function and thus, be used successfully in in vivo studies. However, one major concern that threatens the durability of promising in vivo genome editing therapeutic strategies using Cas9, is the potential for immune rejection of Cas9 expressing edited cells. Preexisting adaptive immunity to Cas9 variant S.aureus, a common human pathogen, has been reported. Therefore, human cells that have been therapeutically treated with SaCas9 are likely to elicit an adaptive memory immune response and trigger killing of Cas9-expressing cells by cytotoxic T-cells. While SaCas9 specific T cells have been identified, the adaptive immune response to SaCas9 has not been fully characterized. The primary human T-cell response to SaCas9 will be readdressed, by probing human serum of healthy and HLA (Human Leukocyte Antigen)-typed human blood donors (Aim 1). In addition, to identify novel and non-immunogenic SaCas9 variants we will conduct a comprehensive mutational screen to elucidate the structure-function relationship of SaCas9. This will lay groundwork for generating a universal SaCas9 that is potentially non-immunogenic for a variety of HLA-types (Aim 2). The proposed work will broaden the knowledge in the gene editing community and expand its implications for future clinical application of CRISPR/Cas in gene therapy.