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- W2922098147 abstract "Purpose Lung transplantation (LTx) is life-saving treatment for end-stage respiratory failure. Our ultimate goal is to improve the outcome of LTx by upregulating the IL-10 gene, an anti-inflammatory and immune down-regulating cytokine, in the whole lung utilizing CRISPR-based technology. The CRISPR/Cas9 system, originally reported as a genome editing system, is being rapidly developed for various applications. CRISPR-mediated gene activation enables us to achieve specific and robust transcriptional activation of an endogenous gene by targeting the genomic DNA. The purpose of this study is to achieve CRISPR-mediated targeted IL-10 gene activation in vitro to generate a viral vector for future whole organ studies. Methods Seven guide RNAs (gRNA) were designed to target the promoter region of IL-10 gene of the rat genome (Figure A). First, each gRNA expressing plasmid was transfected together with a Cas9 activator (dSaCas9-VPR) expressing plasmid to a rat airway epithelial cell line (L2) and lung macrophage cell line (NR8383). Second, a single activation vector, which expressed both gRNA and Cas9 activator, was generated and transfected. The relative expression level of rat IL-10 was measured by qPCR. To determine the optimal viral vector for gene delivery to the lung, different adeno-associated (AAV) virus serotypes were tested in vitro using reporter genes. Results Both epithelial (Figure B) and macrophage cell lines demonstrated enhanced IL-10 gene expression when transfected with Cas9 activator and gRNA4 at 48h. Using a single vector, the gRNA expressing vector significantly increased IL-10 gene expression compared to mock (non-targeted) vectors in epithelial cells (Figure C). In viral vector testing, AAV1 and AAV6 showed effective transduction in macrophage cells. Conclusion Targeted IL-10 gene activation was achieved in both epithelial and macrophage cell lines. CRISPR-mediated IL-10 gene activation of the lung holds considerable promise as a potential gene therapy strategy." @default.
- W2922098147 created "2019-03-22" @default.
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- W2922098147 date "2019-04-01" @default.
- W2922098147 modified "2023-09-27" @default.
- W2922098147 title "CRISPR-Mediated IL-10 Gene Activation as a Novel Gene Therapeutic Strategy in Lung Transplantation" @default.
- W2922098147 doi "https://doi.org/10.1016/j.healun.2019.01.634" @default.
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