Program in Molecular Medicine; RNA Therapeutics Institute; Division of Endocrinology, Metabolism and Diabetes, Department of Medicine; Department of Molecular,Cell and Cancer Biology; Li Weibo Institute for Rare Diseases Research; Graduate School of Biomedical Sciences
Biochemical Phenomena, Metabolism, and Nutrition | Cellular and Molecular Physiology | Endocrine System Diseases | Endocrinology | Hormones, Hormone Substitutes, and Hormone Antagonists | Molecular Biology | Molecular, Cellular, and Tissue Engineering | Nutritional and Metabolic Diseases
Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin [1,2], disturbances in glucose and lipid fluxes [3-5] and comorbidities including steatohepatitis  and cardiovascular disease [7,8]. Despite extensive efforts at prevention and treatment [9,10], diabetes afflicts over 400 million people worldwide . Whole body metabolism is regulated by adipose tissue depots [12-14], which include both lipid-storing white adipocytes and less abundant 'brown' and 'brite/beige' adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health [15-18]. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing [19,20] to enhance 'browning' of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP1 [21,22] with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.
Bioengineering, CRISPR, cell therapy, obesity, Type 2 diabetes, brown adipocytes
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DOI of Published Version
bioRxiv 2020.10.13.337923; doi: https://doi.org/10.1101/2020.10.13.337923. Link to preprint on bioRxiv.
Tsagkaraki E, Nicoloro SM, DeSouza T, Solivan-Rivera J, Desai A, Shen Y, Kelly M, Guilherme AL, Henriques F, Ibraheim R, Amrani N, Luk K, Maitland S, Friedline RH, Tauer L, Hu X, Kim JK, Wolfe SA, Sontheimer EJ, Corvera S, Czech MP. (2020). CRISPR-enhanced human adipocyte 'browning' as cell therapy for metabolic disease [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/2020.10.13.337923. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1814
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