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Pietro Genovese, Ph.D. - "Towards Clinical Translation of Safe and Effective Hematopoietic Stem Cell Gene Editing for the Correction of SCID-X1 "

Gene transfer technologies and new gene therapy strategies, Unit San Raffaele -Telethon Institute for Gene Therapy (SR-TIGET) San Raffaele Scientific Institute IRCCS, Ospedale San Raffaele, Milano
When Jul 04, 2017
from 12:00 PM to 01:30 PM
Where Tigem Auditorium "Vesuvius"
Contact Name
Contact Phone 081-19230659
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Short CV

The scope of genetic engineering of hematopoietic stem/progenitor cells (HSPC) has broadened from gene replacement to genome editing using artificial nucleases, enabling precise modification of endogenous genes. We exploited this strategy on HSPC to insert a functional cDNA into IL2RG gene, whose mutations cause SCID-X1, thus restoring its function and physiologic expression while avoiding the risk of insertional mutagenesis. To support the rationale and explore the safety of gene correction we developed a mouse model carrying a mutated IL2RG gene in place of Il2rg. To evaluate efficacy and safety of hematopoietic reconstitution from a limited number of corrected HSPC we performed competitive transplant with WT and IL2RG-/- HSPC and found that 10% of WT cells fully reconstitute the lymphoid compartments and that administration of a conditioning regimen before HSPC infusion is required to protect from the risk of lymphoma development from the transplanted progenitors. To validate the gene correction strategy in the disease model we developed a gene editing protocol based on CRISPR/Cas9 on murine IL2RG-/- HSPC. Upon transplant, the gene corrected cells were able to generate functional B and T lymphoid lineages, showing a clear selective advantage over uncorrected cells. The corrected cells persisted long-term in the mice and generated a functional T cell response upon in vivo challenge with a pathogen, indicating that IL2RG edited cells are able to partially correct the disease phenotype. Furthermore, by optimizing the gene editing protocol for human HSPC we could attain the threshold of IL2RG editing required for safe and effective disease rescue. By combining donor DNA delivery by AAV6 and advanced generation ZFNs we measured ~35% HDR in the bulk treated CD34+ cells and ~13% HDR upon transplant in NSG mice. Deep sequencing performed on treated CD34+ proved the high specificity of our optimized ZFNs, with no significant modification at any of the off-target sites identified by GUIDE-Seq for earlier generation ZFNs. Finally, to establish a clinical ready protocol, we scaled up the process, treating up to 25 million cells with highly qualified reagents. These studies established safety and robustness of HSPC gene editing for SCID-X1 and will be instrumental for the design of the protocol for its first clinical testing.

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