Genetic engineering of primary human mesenchymal stromal cells using the CRISPR/Cas9 synergistic activation mediator

Background: Genomic editing technologies provide rich opportunities to approach inherited human diseases, but gene delivery strategies are still challenging. While engineered induced pluripotent stem cells or adeno-associated viral vectors may be associated with severe adverse effects, mesenchymal stromal cells (MSC) have abundantly been used for various cell therapeutic strategies and have been proven to be safe in a large number of clinical trials. MSC can be expanded ex vivo on a large scale, and genetic engineering of these somatic stem cells may therefore offer an alternative mode of gene delivery. Methods: We applied the guide RNA-directed CRISPR/Cas9 synergistic activation mediator (SAM) technology in immortalized (iMSC) and primary human MSC (pMSC) in order to activate gene expression of CEBPA and CIITA . Results: After meticulous protocol optimization, both genes were successfully induced in MSC. For CIITA induction, we confirmed expression of the downstream target HLA-DR on mRNA and protein level. Unlike adipogenic, chondrogenic or osteogenic genes, which are naturally induced in MSC upon in vitro differentiation, HLA class II molecules are not constitutively expressed in MSC. Lentiviral and transposon-based delivery strategies were applied, but only with lentiviral transduction of the SAM sequences, CEBPA, CIITA, HLA-DR gene and HLA-DR protein expression were achieved in pMSC. Conclusion: Lentiviral transduction of the guided SAM system was capable to induce transcriptionally silent genes in iMSC and pMSC. The resulting genetically engineered somatic stem cells may be suitable and promising for cell-based therapeutic strategies.