RRC ID 73459
Author Meshitsuka S, Ninomiya R, Nagamura-Inoue T, Okada T, Futami M, Tojo A.
Title CRISPR/Cas9 and AAV mediated insertion of β2 microglobulin-HLA-G fusion gene protects mesenchymal stromal cells from allogeneic rejection and potentiates the use for off-the-shelf cell therapy.
Journal Regen Ther
Abstract INTRODUCTION:Mesenchymal stromal cells (MSCs) hold the potential for application as cellular therapy products; however, there are many problems that need to be addressed before the use in clinical settings, these include the heterogeneity of MSCs, scalability in MSC production, timing and techniques for MSC administration, and engraftment efficiency and persistency of administered MSCs. In this study, problems regarding immune rejection caused by human leukocyte antigen (HLA) mismatches were addressed.
METHODS:Umbilical cord-derived MSCs (UC-MSCs) were gene-edited to avoid allogeneic immunity. The HLA class I expression was abrogated by the knock-out of the beta-2-microglobulin (B2M) gene; instead, the B2M-HLA-G fusion gene was knocked-in using the CRISPR/Cas9 system in combination with adeno-associated virus (AAV).
RESULTS:Cell surface markers on gene-edited UC-MSCs were not different from those on primary UC-MSCs. The gene-edited UC-MSCs also retained the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. B2M gene knock-out alone protected cells from allogeneic T cell immune responses but were vulnerable to NK cells. B2M gene knock-out in combination with B2M-HLA-G knock-in protected cells from both T cells and NK cells. The B2M-HLA-G knock-in MSCs retained a good immunosuppressive ability and the addition of these cells into the mixing lymphocyte reaction showed a significant inhibition of T cell proliferation.
CONCLUSIONS:The results of this study demonstrated the possibility that the CRISPR/Cas9 system combined with AAV can be used to effectively disrupt/introduce any gene into UC-MSCs. Our findings suggest that the gene-edited cell line produced here using this method may have a higher ability to escape the cytotoxic activity of immune cells than primary cells, thereby being more advantageous for long-term graft survival.
Volume 21
Pages 442-452
Published 2022-12-1
DOI 10.1016/j.reth.2022.09.009
PII S2352-3204(22)00095-5
PMID 36313397
PMC PMC9582586
Resource
DNA material CSII-EF-MCS (RDB04378) CSII-CMV-MCS (RDB04377)