Define and minimize the immunogenicity of CRISPR-Cas nucleases

点击翻译按钮获取中文摘要

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.