CRISPR-enhanced human adipocyte 'browning' as cell therapy for metabolic disease

Abstract

Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin1,2, disturbances in glucose and lipid fluxes3-5 and comorbidities including steatohepatitis6 and cardiovascular disease7,8. Despite extensive efforts at prevention and treatment9,10, diabetes afflicts over 400 million people worldwide11. Whole body metabolism is regulated by adipose tissue depots12-14, 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 health15-18. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing19,20 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 NRIP121,22 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.