Xiao Wang1, Avanthi Raghavan1, Tao Chen1, Lyon Qiao1, Yongxian Zhang1, Qiurong Ding2, Kiran Musunuru2. 1. From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.). 2. From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.). kiranmusunuru@gmail.com qrding@sibs.ac.cn.
Abstract
OBJECTIVE: Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology-permanent alteration of genomic DNA sequences-mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. APPROACH AND RESULTS: We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. CONCLUSIONS: This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.
OBJECTIVE: Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology-permanent alteration of genomic DNA sequences-mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. APPROACH AND RESULTS: We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9 genome-editing system to target the humanPCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of humanPCSK9 protein, and minimal off-target mutagenesis. CONCLUSIONS: This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the humanPCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.
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