PMID- 36313397
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20221102
IS  - 2352-3204 (Electronic)
IS  - 2352-3204 (Linking)
VI  - 21
DP  - 2022 Dec
TI  - CRISPR/Cas9 and AAV mediated insertion of beta2 microglobulin-HLA-G fusion gene 
      protects mesenchymal stromal cells from allogeneic rejection and potentiates the 
      use for off-the-shelf cell therapy.
PG  - 442-452
LID - 10.1016/j.reth.2022.09.009 [doi]
AB  - 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.
CI  - (c) 2022 The Japanese Society for Regenerative Medicine. Production and hosting by 
      Elsevier B.V.
FAU - Meshitsuka, Sohsuke
AU  - Meshitsuka S
AD  - Department of Molecular Therapy, Advanced Clinical Research Center, The Institute 
      of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
AD  - Keijinkai Medical Corporation, Tokyo 160-0008, Japan.
FAU - Ninomiya, Ryo
AU  - Ninomiya R
AD  - Department of Molecular Therapy, Advanced Clinical Research Center, The Institute 
      of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
AD  - Daiwa Pharmaceutical Co, Ltd., Tokyo 154-0024, Japan.
FAU - Nagamura-Inoue, Tokiko
AU  - Nagamura-Inoue T
AD  - Department of Cell Processing and Transfusion/Laboratory Medicine, IMSUT 
      Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo 
      108-8639, Japan.
FAU - Okada, Takashi
AU  - Okada T
AD  - Division of Molecular and Medical Genetics, The Institute of Medical Science, The 
      University of Tokyo, Tokyo 108-8639, Japan.
FAU - Futami, Muneyoshi
AU  - Futami M
AD  - Department of Molecular Therapy, Advanced Clinical Research Center, The Institute 
      of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
AD  - Project Division of Innovative Diagnostics Technology Platform, The Institute of 
      Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
FAU - Tojo, Arinobu
AU  - Tojo A
AD  - Department of Molecular Therapy, Advanced Clinical Research Center, The Institute 
      of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
AD  - Institute of Innovation Advancement, Tokyo Medical and Dental University, 
      113-8510, Japan.
LA  - eng
PT  - Journal Article
DEP - 20221014
PL  - Netherlands
TA  - Regen Ther
JT  - Regenerative therapy
JID - 101709085
PMC - PMC9582586
OTO - NOTNLM
OT  - AAV
OT  - AAV, adeno-associated virus
OT  - Allogenic rejection
OT  - CRISPR/Cas9
OT  - FASL, FAS ligand
OT  - GVHD, graft versus host disease
OT  - HLA, human leukocyte antigen
OT  - HLA-G
OT  - HR, homologous recombination
OT  - HSC, hematopoietic stem cells
OT  - ITR, inverted terminal repeats
OT  - KIR, killer-cell immunoglobulin-like receptors
OT  - LILR, leukocyte immunoglobulin-like receptors
OT  - MLR, mixed lymphocyte reaction
OT  - MSC, mesenchymal stromal cells
OT  - PBMC, peripheral blood mononuclear cells
OT  - PS, penicillin-streptomycin
OT  - SD, standard deviation
OT  - UC-MSCs
COIS- RN is employed by Daiwa Pharmaceutical. SM is employed by Keijinkai Medical 
      Corporation. AT received a research grant from Daiwa Pharmaceutical. TN-I, TO, 
      and MF has no conflicts of interest.
EDAT- 2022/11/01 06:00
MHDA- 2022/11/01 06:01
PMCR- 2022/10/14
CRDT- 2022/10/31 04:56
PHST- 2022/08/20 00:00 [received]
PHST- 2022/09/14 00:00 [revised]
PHST- 2022/09/25 00:00 [accepted]
PHST- 2022/10/31 04:56 [entrez]
PHST- 2022/11/01 06:00 [pubmed]
PHST- 2022/11/01 06:01 [medline]
PHST- 2022/10/14 00:00 [pmc-release]
AID - S2352-3204(22)00095-5 [pii]
AID - 10.1016/j.reth.2022.09.009 [doi]
PST - epublish
SO  - Regen Ther. 2022 Oct 14;21:442-452. doi: 10.1016/j.reth.2022.09.009. eCollection 
      2022 Dec.