Therapeutic Genome Editing and its Potential Enhancement through CRISPR Guide RNA Modifications

Hematopoietic stem cell transplant (HSCT) is a treatment option for monogenic diseases of the immune system. While allogeneic HSCT can be curative for these diseases, there remain significant limitations to this approach such as graft-versus host disease and graft rejection. Autologous gene therapy using viral vectors containing a corrective transgene is being developed for some of these disorders, most successfully for adenosine deaminase deficiency. However, for other immune disorders, the transgene needs to be expressed in a precise, developmental and lineage specific manner to achieve functional gene correction and to avoid the risks of cellular transformation. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards using genome editing by homologous recombination (HR) to correct a disease-causing mutation by precisely modifying the genome of hematopoietic stem cells. CRISPR/Cas-mediated genome editing relies on guide RNAs to direct site-specific DNA cleavage mediated by the Cas endonuclease. We chemically synthesized single guide RNAs (sgRNAs) targeting three different genes (IL2RG, HBB, and CCR5), alongside sgRNA variants containing modified nucleotides. We show that sgRNAs containing modified nucleotides dramatically enhance genome editing compared to unmodified sgRNAs. When co-delivering chemically modified sgRNAs and Cas9 mRNA we observe ~70% in/del frequencies in human primary T cells, and ~40% in hematopoietic stem and progenitor cells. In addition, we present a CRISPR/Cas9 gene editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve HR in hematopoietic stem cells. Collectively, these studies outline a CRISPR-based methodology for targeting hematopoietic stem cells by HR to advance the development of next-generation therapies for monogenic disease of the immune system.