Immunodeficiency: primary or acquired
Homozygous loss-of-function mutations in IL-10 and IL-10-receptors (IL-10R) cause severe infantile inflammatory bowel disease. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure. However, limited donor availability and morbidity/mortality prevent the wide use of allo-HSCT. An approach based on gene-correction of patients derived hematopoietic stem and progenitor cells (HSPCs) will allow us to use an autologous HSCT. We have developed CRISPR/Cas9-based strategy to target the IL-10 locus. We selected 3 small guide RNAs (sgRNAs). To test the targeted integration mediated by those guides, we designed recombinant AAV6 (rAAV6) DNA donor templates. We observed for IL10-3, -5 and -6 an average integration of 20%, 45% and 55% in CD34+; while 17%, 28% and 30% in CD4+ T cells. Based on these data, we selected the IL10-6 guide for further studies. We designed rAAV6 carrying a codon optimized IL-10 cDNA. Digital droplet PCR showed 38% of integration at IL-10 locus in CD34+ cells. We are currently testing sgRNAs targeting IL-10RA locus.
As the regulation of IL-10 is still poorly understood, we will use this technology to define molecular signals sustaining normal IL-10 production by Tr1 cells. To this goal, we generated Tr1 clones which produce high level of IL-10 and inhibit proliferation of CD4+ responder T cells.
In conclusion, we identified a sgRNA mediating high targeted integration at the IL-10 locus in both human HSPCs and T cells. These tools will be used to gene correct IL-10 deficient cells and for studying IL-10 regulation and the molecular mechanisms underlying IL-10 deficiency.