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2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 Goldstein JM, Tabebordbar M, Zhu K, Wang LD, Messemer KA, Peacker B, Ashrafi Kakhki S, Gonzalez-Celeiro M, Shwartz Y, Cheng JKW, Xiao R, Barungi T, Albright C, Hsu YC, Vandenberghe LH, Wagers AJ. 2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Skeletal muscle is a highly regenerative tissue, but muscle repair potential is increasingly compromised with advancing age. In this study, we demonstrate that increased NF-κB activity in aged muscle fibers contributes to diminished myogenic potential of their associated satellite cells. We further examine the impact of genetic modulation of NF-κB signaling in muscle satellite cells or myofibers on recovery after damage. These studies reveal that NF-κB activity in differentiated myofibers is sufficient to drive dysfunction of muscle regenerative cells via cell-non-autonomous mechanisms. Inhibition of NF-κB, or its downstream target Phospholipase A2, in myofibers rescued muscle regenerative potential in aged muscle. Moreover, systemic administration of sodium salicylate, an FDA-approved NF-κB inhibitor, decreased inflammatory gene expression and improved repair in aged muscle. Together, these studies identify a unique NF-κB regulated, non-cell autonomous mechanism by which stem cell function is linked to lipid signaling and homeostasis, and provide important new targets to stimulate muscle repair in aged individuals. -
Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Array© 2015 American Heart Association, Inc. -
Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics. -
Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.Copyright © 2016, American Association for the Advancement of Science.