The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.

Agonists of the G protein-coupled receptor TGR5 have long been sought-after for their metabolic benefits. Intestinal TGR5 activation induces secretion of the antidiabetic hormone GLP-1, which can systemically improve diabetes phenotypes in multiple organs. However, no TGR5 agonist drug candidate has succeeded in clinical trials due to their low potency and unwanted side effects. A challenge in the field has been the development of TGR5 agonists that are non-toxic, long-acting, and have functional selectivity for G protein-biased agonism. In this study, we propose a systematic pipeline for engineering optimal TGR5 agonists with antidiabetic properties. This pipeline is interdisciplinary, combining in silico, in vitro, and in vivo assays to design and validate drug candidates. We identify 2 lead compounds that outline the necessary beneficial properties for a successful TGR5 agonist against diabetes. We uncover the molecular mechanisms that allow TGR5 agonists to induce the transcription of their target, TGR5, in intestinal enteroendocrine cells. Lastly, we investigate the molecular interactions of our lead candidates in the TGR5 binding pocket to identify optimal parameters for stability and biological activity. Our strategy for TGR5 agonist design and evaluation has the potential to guide the discovery process for targeted TGR5 therapeutics for metabolic diseases.