Tu217 - A Humanized Mouse Model of VEO-IBD by crispr/cas9 Editing of the IL10 Gene in Human Hspcs

Rosa Romano; Esmond Lee; Liwen Xu; Robert Freeborn; Bing Wang; Neetu Saini; Suzette Shipp; Molly Uyeda; Pauline Chen; Mansi Narula; Mara Pavel-Dinu; Matthew Porteus; Rosa Bacchetta; Maria Grazia Roncarolo

Abstract Text: Very early onset inflammatory bowel disease (VEO-IBD) is a severe form of IBD that affects children younger than 6-years-old. In VEO-IBD, uncontrollable immune responses to gut microbiome-derived antigens result in excessive and chronic inflammation of the intestines. The most common and severe form of VEO-IBD is caused by loss-of-function (LOF) mutations of the IL10, IL10RA, and IL10RB genes, which encode the immunoregulatory cytokine IL-10 or its receptor subunits. Although Il10-deficient mice provided critical insights in IBD, differences in IL-10 biology and IBD pathophysiology between mouse and human warrant the need for a humanized mouse model of VEO-IBD. Therefore, we designed a multi-guide CRISPR/Cas9 gene editing strategy in which the IL10 locus of human CD34+ hematopoietic stem and progenitor cells (HSPCs) is disrupted with high efficiency and negligible off-target events. As a control, rAAV-mediated knock-in (KI) of wildtype IL10 cDNA at the endogenous IL10 locus of these IL10-knock-out (KO) HSPCs was achieved with high integration efficiency. To create a humanized VEO-IBD mouse model, we transplanted specific-pathogen-free (SPF) NSG-SGM3 mice with wild-type and IL10-KO HSPCs and observed high reconstitution of the human immune system in multiple tissues including spleen, bone marrow, and mesenteric lymph nodes. However, we did not observe colitis in mice transplanted with IL10-KO HSPCs which was comparable to germ-free Il10-deficient mice. Therefore, we aim to challenge these mice with IBD-triggering bacteria in follow-up cohorts. In conclusion, the successful reconstitution of mice with human IL10-deficient HSPCs will, upon further optimization, allow the development of a humanized mouse model of VEO-IBD.