Employing novel humanized murine strains to develop hematopoietic stem cell gene therapeutic approaches for very early-onset inflammatory bowel disease due to IL10-receptor deficiency.

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Loss-of-function mutations in interleukin-10 (IL10) or the IL10 receptor (IL10R) lead to a life-threatening immune deficiency presenting as severe very early-onset inflammatory bowel disease (VEO-IBD). IL10 is an immunosuppressive cytokine that is secreted by a variety of hematopoietic cells, primarily myeloid, B and T cells, which reduces inflammation, at least in part by limiting the secretion of pro-inflammatory cytokines. The receptor for IL10 is composed of two IL10R1 and two IL10R2 chains and is expressed predominantly on hematopoietic cells. Mice deficient for either IL10R or IL10 develop spontaneous colitis and similarly, patients with loss-of-function mutations of either the IL10RA or IL10RB develop severe VEO-IBD. Recent studies have shown that hematopoietic stem cell (HSC) transplantation (HSCT) may lead to a sustained remission of the disease, but allogeneic HSCT carries significant risks of acute toxicities and graft-versus-host-disease (GVHD) and requires a suitable HLA-matched donor. Preliminary data from our group has indicated that Il10rb gene transfer into IL10Rβ-deficient bone marrow precursors followed by HSCT into IL10Rβ knockout models can prevent the development of IBD. We propose to use gene transfer into autologous HSCs via lentivirus vectors (permitting either ubiquitous or lineage-specific Il10rb expression) to restore IL10R signaling and provide an alternative treatment option. In this proposal, we will develop and optimize lentivirus vectors to express Il10rb, determine the in vitro and in vivo efficacy and safety of these vectors in established murine knockout models. In addition, we will utilize novel humanized mouse models recently developed that permits the use of patient derived CD34+ HSCs to assess both the functional consequence of IL10R2 deficiency in human cells in vivo as well as the effectiveness of gene transfer in patient-derived HSCs. Moreover, we plan to generate CRISPR- Cas9 gene-edited HSCs derived from IL10R1- and IL10R2-deficient human patients and assess the frequency and precision of editing events and functional consequences. These investigations will provide the preclinical data clarifying the transgenic expression levels needed for correcting the immune dysregulation and the safety of expressing a signaling molecule in hematopoietic cells. Finally, we will determine in both standard IL10Rβ- deficient murine as well as humanized mouse models, transplanted with murine- and patient-derived IL10Rβ/IL10R2-deficient HSCs with lineage-specific Il10rb/IL10RB-expressing lentivirus vectors, the precise cell populations that require IL10Rβ expression to correct immune dysfunction. These studies will enhance our understanding of the basic cellular pathobiology of this rare disease and also address the complexities associated with heterologous expression of transgene in specific components of the hematopoietic compartment. Deep expertise in gene therapy (Williams) and immunology/IBD (Snapper) will form an excellent basis to develop a gene therapy approach for this indication.