Development of a baculoviral CRISPR/Cas9 vector system for beta-2-microglobulin knockout in human pluripotent stem cells.
Mol Genet Genomics
; 299(1): 74, 2024 Aug 01.
Article
em En
| MEDLINE
| ID: mdl-39085666
ABSTRACT
Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Diferenciação Celular
/
Baculoviridae
/
Microglobulina beta-2
/
Células-Tronco Pluripotentes
/
Sistemas CRISPR-Cas
/
Edição de Genes
/
Vetores Genéticos
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Genet Genomics
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China