Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1

Abstract

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

“…Reductions in gene editing rates from in vitro to in vivo studies have been reported by other groups using similar gene editing reagents in hematopoietic stem cells. (Genovese et al , 2014; Hoban et al , 2015; De Ravin et al , 2016; Schiroli et al , 2017) Selection methods using reporter genes may be able to overcome the problem(Dever et al , 2016), but this method has not been shown to be scalable to human trials of gene editing due a significant loss of HSC numbers. However, despite the reduced editing rates in vivo, it is likely that in XHIM a proportionally small number of gene-corrected T cells will be sufficient to allow enough class-switching to significantly ameliorate or cure the disease.…”

Thymus autonomy as a prelude to leukemia

“…In that study, although the cells that originated the malignancy were corrected (transduced) for the γc‐deficiency, the leukemic cells had a transcription pattern identical to normal wild‐type, nontransduced T lymphocytes, suggestive that genotoxicity did not occur . The work from Naldini's laboratory elegantly explored several additional aspects that must be taken into account when attempting to correct the γ c genetic defect by gene therapy . This group generated their own mouse model of SCID‐X1, mimicking a null mutation from patients.…”

Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives

“…Today, this is most often done by 6 of 12 EMBO Molecular Medicine 11: e9958 | 2019 ª 2019 The Author electroporating purified nuclease ribonucleoprotein complexes (i.e., Cas nucleases preassembled with single guide RNA) or nucleaseencoding mRNAs (for the other platforms), followed, when required, by the delivery of the repair template through a viral vector, such as adeno-associated virus-derived (AAV6) or integrase-defective LV (IDLV) (Genovese et al, 2014;Wang et al, 2015;Dever et al, 2016;De Ravin et al, 2016aSchiroli et al, 2017). Today, this is most often done by 6 of 12 EMBO Molecular Medicine 11: e9958 | 2019 ª 2019 The Author electroporating purified nuclease ribonucleoprotein complexes (i.e., Cas nucleases preassembled with single guide RNA) or nucleaseencoding mRNAs (for the other platforms), followed, when required, by the delivery of the repair template through a viral vector, such as adeno-associated virus-derived (AAV6) or integrase-defective LV (IDLV) (Genovese et al, 2014;Wang et al, 2015;Dever et al, 2016;De Ravin et al, 2016aSchiroli et al, 2017).…”

“…Today, this is most often done by 6 of 12 EMBO Molecular Medicine 11: e9958 | 2019 ª 2019 The Author electroporating purified nuclease ribonucleoprotein complexes (i.e., Cas nucleases preassembled with single guide RNA) or nucleaseencoding mRNAs (for the other platforms), followed, when required, by the delivery of the repair template through a viral vector, such as adeno-associated virus-derived (AAV6) or integrase-defective LV (IDLV) (Genovese et al, 2014;Wang et al, 2015;Dever et al, 2016;De Ravin et al, 2016aSchiroli et al, 2017). We and others could overcome at least in part these barriers by extending culture in conditions that drive even the most primitive cells into replication while preserving their engraftment capacity and by tailoring the gene editing machinery to avoid triggering innate immune cellular responses (Genovese et al, 2014;Wang et al, 2015;De Ravin et al, 2016aDever et al, 2016;Schiroli et al, 2017). All these approaches have the advantage to achieve a high but transient spike of nuclease expression in the cultured cells, thus limiting its activity to a small window of time and, consequently, alleviating toxicity and off-target activity.…”

“…Whereas AAV6 and IDLV can shuttle relatively large DNA molecules into the nucleus, thus enabling large edits and insertions, co-electroporation of short single-or double-stranded oligonucleotides with the nuclease can instead be used for editing few base pairs. Whereas these levels of editing may be sufficient to treat some PID, such as SCID-X1, in which a relatively small fraction of functional progenitors in the administered cell product nearly completely rescued the T-cell deficiency in mouse models and in early gene therapy trials performed with relatively inefficient c-retroviral vectors (Fischer et al, 2015;Schiroli et al, 2017), they may be limiting in other applications, where more robust correction is needed, particularly of long-term repopulating HSC. The major hurdle to exploit targeted gene editing in HSC is that procedures based on HDR suffer from limited efficiency in the most primitive progenitors, likely due to low expression and proficiency of the HDR machinery, cell quiescence, and limited uptake of repair template (Genovese et al, 2014).…”