(500d) Reprogramming MHC Specificity of Immune Cells By Crispr-Cas9 Assisted Cassette Exchange
AIChE Annual Meeting
2016 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Synthetic Biology Applications
Wednesday, November 16, 2016 - 9:24am to 9:42am
Allogeneic cellular transplantation is widely employed to treat various genetic diseases and hematological malignancies. Locating suitable donors for these procedures is often challenging, as MHC/HLA gene alleles need to be matched in order to prevent transplant rejection. We report here a proof of concept study for an ex-vivo CRISPR-Cas9-based approach for the precise exchange of MHC alleles (~5kb) at the native genomic locus. For initial evaluation, the method was performed on immortalized mouse antigen-presenting cells (RAW264.7 macrophages) expressing high levels of surface MHC (H2-Kd). Genomic exchange at the H2-K locus was achieved via co-transfection of cells with a plasmid carrying Cas9 and guide RNA targeting the H2-Kd locus and a homology donor repair template encoding an alternate MHC allele (H2-Kb). Following homology directed repair, modified cells were isolated by flow cytometry based on expression of new MHC. Engineered cells expressing alternate H2-Kb allele were able to present a model antigen peptide (OVA-SINFEKLL) and activate a cognate T cell hybridoma line, demonstrating they were active for downstream immune functions. We envision this approach could be used in the future to improve MHC/HLA matching in cellular transplantation.