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Sci Rep ; 12(1): 13202, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35915300

ABSTRACT

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system represents a powerful gene-editing tool and could enable treatment of blinding diseases of the retina. As a peptide of bacterial origin, we investigated the immunogenic potential of Cas9 in models of retinal immunocompetent cells: human microglia (IMhu) and ARPE-19 cells. Transfection with Streptococcus pyogenes-Cas9 expression plasmids (SpCas9 plasmid) induced Cas9 protein expression in both cell lines. However, only ARPE-19 cells, not IMhu cells, responded with pro-inflammatory immune responses as evidenced by the upregulation of IL-8, IL-6, and the cellular activation markers HLA-ABC and CD54 (ICAM). These pro-inflammatory responses were also induced through transfection with equally sized non-coding control plasmids. Moreover, viability rates of ARPE-19 cells were reduced after transfection with both the SpCas9 plasmids and the control plasmids. Although these results demonstrate cell type-specific responses to the DNA plasmid vector, they show no evidence of an immunogenic effect due to the presence of Cas9 in models of human retinal pigment epithelial and microglia cells. These findings add another layer of confidence in the immunological safety of potential future Cas9-mediated retinal gene therapies.


Subject(s)
Bacterial Proteins , CRISPR-Cas Systems , Bacterial Proteins/metabolism , Epithelial Cells/metabolism , Gene Editing/methods , Humans , Immunity , Plasmids/genetics , Retinal Pigments
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