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      In vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency

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          Abstract

          <p class="first" id="d6793323e382">CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with alpha-1 antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model. Specifically, we co-injected two AAVs: one expressing Cas9 and another encoding an AAT guide RNA and homology-directed repair template. In both neonatal and adult mice, this treatment partially restored M-AAT in the serum. Furthermore, deep sequencing confirmed both indel mutations and precise gene correction in the liver, permitting careful analysis of gene editing events <i>in vivo</i>. This study demonstrates a proof of concept for the application of CRISPR-Cas9 technology to correct AAT mutations <i>in vivo</i> and validates continued exploration of this approach for the treatment of patients with AAT deficiency. </p>

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          Most cited references23

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          Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.

          Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.

            We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
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              CRISPR-mediated direct mutation of cancer genes in the mouse liver

              The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells 1 . Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) 2–4 to the liver and directly target the tumor suppressor genes Pten 5 and p53 6 , alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology 7, 8 . Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
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                Author and article information

                Journal
                Human Gene Therapy
                Human Gene Therapy
                Mary Ann Liebert Inc
                1043-0342
                1557-7422
                May 14 2018
                May 14 2018
                Affiliations
                [1 ]RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
                [2 ]Horae Gene Therapy Center and Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts.
                [3 ]Departments of Pediatrics and Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts.
                [4 ]Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, China.
                [5 ]Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
                [6 ]Medical Research Institute, Wuhan University, Wuhan, China.
                [7 ]Program in Molecular Medicine and Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
                Article
                10.1089/hum.2017.225
                6110121
                29597895
                1d21252d-c133-43ef-93c8-db147c10d50b
                © 2018
                History

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