Hematopoietic stem cells (HSC) have long been a preferred target for ex vivo gene therapy. The ease of collecting HSC, their ability to survive in ex vivo culture, and capacity to home and engraft upon transplant are important features that support HSC use in gene therapy. The scope of genetic engineering has recently broadened from gene replacement to targeted gene editing using engineered nucleases, which enable precise sequence modification of a locus of interest. When working with the group of Prof. Luigi Naldini, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), we provided a major contribution this field by developing the first effective gene editing strategy for targeted gene addition or in situ correction of inherited mutations in human hematopoietic stem cells. By tailoring culture conditions and gene delivery vehicles, we overcome the biologic barriers that specifically constrain gene targeting in the most primitive subset of hematopoietic progenitors and developed a protocol that allows targeted integration of a transgene expression cassette into a “safe harbor” site or direct correction of the IL2RG gene of HSCs from healthy donors and X-linked severe combined immunodeficiency (SCID-X1) patients (Genovese et al., Nature 2014). This work was the first poof that targeted gene modifications can be efficiently obtained in HSC that preserve their repopulation potential and formed the basis for several other studies in the field, some of which will shortly enter clinical testing.
Despite recent advance in this field, several hurdles need to be solved before we can fully benefit from the predicted safety and precision of genetic engineering afforded by these new technologies. A major issue is that gene editing in primitive HSC is constrained by gene transfer efficiency and limited proficiency of homology directed DNA repair (HDR), likely due to HSC quiescence and low expression of the DNA repair machinery. Moreover, the induction of DNA double strand breaks (DSB) required for initiating the GE process may per se trigger apoptosis, differentiation, cell senescence and replicative arrest, thus exacerbating the risk of exhaustion or limiting their engraftment capacity. Recently, we assess the impact of this editing procedure on the treated HSC by performing an unbiased single-cell transcriptomic analysis (Schiroli, Conti et al., Cell Stem Cell 2019). These studies uncovered cumulative activation of P53 dependent DNA Damage Response (DDR), which receives multiple converging inputs during the editing procedure, including sensing of nuclease induced DNA double strand brakes and of AAV6 proviruses used for DNA template delivery. However, we found that this functional impairment could be overcome by short transitory dampening of DDR during the editing procedure achieved thought the delivery of mRNA encoding for a p53 inhibitor peptide (GSE56).
Since this DDR response decreases the output of HSC editing, we transiently co-delivered during electroporation different factors that counteract this response. By this screening, we identified an adenoviral protein (E4orf6/7) that forced cell cycle progression, thus boosting homology-mediated editing and increasing the yield of edited HSPC in xeno-transplanted NSG mice. To assess clonality of the edited HSC, we developed a barcoding-based strategy that allows monitoring clonal behavior and hematopoietic graft composition after HDR mediated editing (BAR-seq). By this strategy, we proved polyclonal reconstitution and preserved self-renewal and multi-potency of individual HSC edited with our optimized protocols (Ferrari, Jacob et al., Nature Biotechnology 2020). These findings provide molecular evidence of the feasibility of seamless targeted gene editing in HSPC able to polyclonal, long-term engraftment, thus giving confidence to its prospective translation.
Our BAR-seq bioinformatics pipeline is freely available here with an user friendly platform thanks to the BioInfoTiget group (Stefano Beretta and Ivan Merelli). A detailed protocol for HSPC editing and clonal tracking (Ferrari et al., Nature Protocol 2021) is available here.
Because most therapeutic applications would require substantial amounts of gene edited HSC, it will be crucial to further enhance the HDR efficiency while at the same time fully preserving their long-term repopulating activity. Our lab actively tackle this challenge by developing, testing and implementing innovative approaches aimed to broaden the therapeutic potential of these innovative technologies.
Because most therapeutic applications would require substantial amounts of gene edited HSC, it will be crucial to further enhance the HDR efficiency while at the same time fully preserving their long-term repopulating activity. Our lab actively tackle this challenge by developing, testing and implementing innovative approaches aimed to broaden the therapeutic potential of these innovative technologies.