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

Obesity and type 2 diabetes are associated with disturbances in insulin regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as 'brown' and 'brite/beige' adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of immortalized human brown adipocytes into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary beige adipocytes. Here we applied methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupted the thermogenic suppressor gene NRIP1 by CRISPR. SpyCas9 protein/sgRNA complexes delivered ex vivo were fully degraded by the human cells following high efficiency NRIP1KO that markedly elevated the brown-like gene expression profile with no detectable off target editing. Implantation of such 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 enhancement of human adipocyte that does not expose the recipient to immunogenic Cas9 or delivery vectors.

肥胖与2型糖尿病均与胰岛素调控的葡萄糖及脂质代谢流紊乱相关，且会引发包括心血管疾病、脂肪性肝炎在内的多种严重并发症。全身代谢由储存脂质的白色脂肪细胞，以及表达产热解偶联蛋白1（UCP1）并分泌有益于代谢健康的细胞因子的棕色脂肪细胞与亮米色（brite/beige）脂肪细胞共同调控。将永生化人源棕色脂肪细胞移植至肥胖小鼠体内可改善其糖耐量，但由于原代米色脂肪细胞丰度极低，该方法向临床转化的进程受到阻碍。本研究通过相关方法，从少量人体皮下脂肪组织样本中大量扩增人源脂肪细胞祖细胞，随后利用CRISPR技术敲除产热抑制基因NRIP1。体外递送的SpyCas9蛋白/sgRNA复合物可在高效完成NRIP1敲除（NRIP1KO）后被人源细胞完全降解，该过程可显著上调棕色样基因表达谱，且未检测到脱靶编辑现象。将此类经CRISPR增强的人源或小鼠"棕色样"脂肪细胞移植至高脂饮食喂养的小鼠体内，相较于移植未修饰脂肪细胞的对照组小鼠，可降低其体脂含量与肝脏甘油三酯水平，并改善糖耐量。本研究成果为通过基于CRISPR的基因增强技术改造人源脂肪细胞以改善代谢稳态提供了一种治疗策略，该策略不会使受体暴露于具有免疫原性的Cas9蛋白或递送载体中。