TALEN-based gene editing offers hope for patients with sickle cell disease
Sickle cell disease (SCD), a condition originating from a single A > T point mutation in the HBB gene coding for hemoglobin, is one of the most common inherited disorders worldwide. However, the only available cure for SCD is allogeneic hematopoietic stem cell transplantation (allo-HSCT), a procedure with substantial rates of morbidity and mortality. Ex vivo nuclease-mediated HBB correction of autologous hematopoietic stem and progenitor cells (HSPCs) represent a promising alternative to allo-HSCT, provided various challenges are addressed. These include avoiding the introduction of inactivated HBB alleles, which reduces the proportion of corrected cells in the final product, and preventing activation of p53 pathways, limiting engraftment capacity. A preclinical proof of concept, based on TALEN gene editing and non-viral DNA delivery, circumvents these issues and has the potential to be further developed for therapeutic application.
Viral and non-viral mediated DNA delivery achieve comparable HBB correction in vitro
Two TALENs were designed for this study: TALEN-HBBss and TALEN-HBBββ, which are respectively specific for the mutant and wild-type versions of HBB exon 1. The TALEN mRNAs were produced by TriLink. To compare the gene editing efficiency obtained with viral (adeno-associated virus 6) and non-viral (single-stranded oligonucleotide) DNA delivery strategies, the TALENs were administered in combination with DNA repair templates containing the sickle-to-wild type mutation to mobilized HSPCs from healthy donors or homozygous hbSS patients. HBB gene correction was assessed using digital droplet PCR (ddPCR) or AmpliconSeq to determine the HDR/indel ratio. Following optimization of the TALEN-mediated editing protocol, both viral and non-viral mediated DNA delivery achieved highly efficient gene correction in clinically relevant HSPCs, while mitigating the risk of frequent HBB indels.
Non-viral DNA delivery provides higher engraftment and gene correction
To establish whether the viral and non-viral DNA delivery strategies were capable of editing long-term hematopoietic stem cells, plerixafor (PLX)-mobilized HSPCs from three healthy donors were edited prior to transplantation into immunodeficient NCG mice. 16 weeks post-transplant, human chimerism was measured by flow cytometry and the HDR frequency was assessed with ddPCR. Non-viral mediated DNA delivery was shown to outperform viral-mediated editing in terms of both engraftment and HDR efficiency.
Non-viral DNA delivery mitigates P53 activation
Single-cell RNA sequencing (scRNAseq) on the PLX-HSPCs used for transplantation indicated that the superior engraftment seen with non-viral mediated DNA delivery could be due to a higher proportion of primitive HSPCs being maintained with this method compared to viral DNA delivery. scRNAseq additionally revealed less activation of the p53 pathway with the non-viral DNA delivery strategy, suggesting this approach to better preserve HSPC fitness.
Conclusion
Coupling TALEN gene editing with non-viral DNA delivery allows for correcting the single-point mutation underlying SCD pathophysiology. The comprehensive preclinical dataset generated by Moiani et al. sets the stage for future clinical trials, in which the strategy conferring the greatest therapeutic benefit for SCD should be determined.
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Article reference: Moiani A., Letort G., Lizot S., et al. Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells. Nat Commun. 2024;15(1):4965. https://www.nature.com/articles/s41467-024-49353-3