Category: 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.
Rosa Romano– PhD, Stanford University
Rosa Pavel-Dinu– PhD, Stanford University
Rosa Bacchetta– Associate Professor, Department of Pediatrics, Human Gene Therapy at the Lucile Salter Packard Children’s Hospital, Stanford school of medicine, Stanford, CA, USA
Matthew Porteus– Professor, Stanford University
Maria Grazia Roncarolo– Professor, Stanford University