37. Towards Clinical Translation of Hematopoietic Stem Cell Gene Editing for the Correction of SCID-X1 Mutations

Abstract

The scope of genetic engineering of hematopoietic stem/progenitor cells (HSPC) has broadened from gene replacement to targeted genome editing using a choice of artificial nucleases, which enables precise modifications of endogenous genes. We recently showed that gene editing in the primitive HSPC is constrained by gene transfer efficiency and limited proficiency of homology directed DNA repair. By combining Integrase Defective Lentiviral Vectors (IDLV) for donor template delivery and mRNA transfection for nucleases expression and tailoring culture conditions, we overcame in part these barriers and provided evidence of increased targeted integration (TI) in human HSPC. We achieved TI of a corrective cDNA into a mutational hotspot of IL2RG gene in long-term repopulating HSPC with efficiency and specificity potentially suitable for clinical translation. Here, in order to improve the tolerability of the procedure and establish a transferable-to-the-clinic gene correction protocol we optimized reagents and scaled-up the gene editing procedure. We developed new ZFNs targeting the upstream region of the IL2RG gene to correct the majority of SCID-X1 mutations with only one ZFN/donor set. By targeting a corrective cDNA into intron-1 of IL2RG gene in primary T cells, we found that targeted cells were functionally indistinguishable from wild-type ones, proving the functionality of the edited gene. We compared the performance of IDLV and AAV6 as donor vehicles for HSPC targeting and found that both vectors are similarly proficient in the delivery of donor templates for HDR, allowing up to 20% TI in bulk HSPC. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we included modified nucleotides during mRNA production and performed HPLC purification after in-vitro transcription. The use of this optimized mRNA and clinical grade purification of IDLV (based on DNA removal followed by anion exchange chromatography) allowed decreasing type-1 interferon activation and electroporation toxicity, respectively. To further optimize ex-vivo HSPC manipulation we tested pyrimidoindole derivatives added to the culture and found a combination promoting HSPC expansion in conditions that preserve their primitive phenotype, increasing the yield of edited cells that are able to repopulate NSG mice. Finally, we demonstrated the therapeutic potential of our strategy by correcting the IL2RG gene in HSPC from a genotyped SCID-X1 patient. Currently, we are processing large scale lots of gene corrected cells using a high volume electroporator and have successfully treated up to 40 million of HSPCs. We envisage early clinical translation of this approach, which may benefit SCID-X1 patients while avoiding the risks of allogeneic transplantation or random transgene integration mediated by HSPC gene therapy with conventional integrating vectors.