HEMATOPOIETIC STEM CELL GENE THERAPY AS A NOVEL THERAPEUTIC APPROACH FOR SEVERE CROHN’S DISEASE ASSOCIATED WITH NOD2-DEFICIENCY

Abstract

Abstract Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD). Mounting evidence links NOD2 deficiency with poor clinical outcome, particularly in pediatric and early onset CD. CD patients with pathogenic NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated as a driver of CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stem cell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile (IL-8, TNFa, IL-6, CXCL1/2 and IL-10). In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors. Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in Nod2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, Nod2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated immune markers. Transplanted Nod2KO mice display normal hematopoiesis and stable vector copy numbers in hematopoietic cells. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells (Figure 1). These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.