Generation of CCR5ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immunemediated Diseases

CC chemokine receptor type 5 (CCR5) is a receptor for some proinflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immunemediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived human induced pluripotent stem cells (PMSChiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, offtarget sites were evaluated by the #x201C;Loss of a primer binding site #x201D; technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSCiPSCs, the program of A033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The #x201C;Loss of a primer binding site #x201D; technique showed that no exonic offtarget mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSChiPSCs without detectable offtarget damage. This approach can provide a stable supply of autologous/allogeneic CCR5disrupted PMSChiPSCs that might be a feasible approach for the treatment of immunemediated diseases.