CRISPR-SaCas9-KKH targeting of miR-21 through a single adeno-associated virus vector prolongs survival of GB-bearing mice

Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21) is a known oncogene, abundantly expressed in many cancer types. MiR-21 promotes glioblastoma progression, and lack of miR-21 reduces tumorigenic potential of tumor. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting miR-21 using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic glioblastoma model. The SaCas9-KKH induced a genomic deletion, resulting in lowered miR-21 levels in the brain, leading to reduced tumor growth and improved overall survival. In this study we demonstrated that a single AAV-mediated mir-21 disruption influences glioma development resulting in beneficial anti-tumor outcomes in GB-bearing mice. Overall design: To investigate the therapeutic effect of targeting microRNA-21 in a glioma context we peformed Next Generation Sequencing analysis to confirm gene-editing of the targeted microRNA-21. A wild-type (WT) non-edited cell line and/or SaCas9 only condition were the controls. 1) Co-transfected in vitro in glioma cell lines (CT2A and GL261) with SaCas9 and sgRNA to knockdown miR-21. 2) Tested a single construct containing both SaCas9 and sgRNA to target miR-21 in vitro cell lines. 3) designed a single AAV vector to CRISPR-edit microRNA-21 in vivo in a glioblastoma mouse model. The AAV was delivered locally by injecting it intratumorally in the mice brain. We then analysed FACS sorted tumor tissue for CRISPR-editing of microRNA-21 by Next Generation Sequencing. 4) We screened 5 potential off-targets in the mouse genome of the specific sgRNA we used in this study by Next Generation Sequencing.