Gene correction for SCID-X1 in long-term hematopoietic stem cells

Abstract

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34 + HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

“…Genome editing by combining ribonucleoprotein (RNP, Cas9 protein complexed to synthetic stabilized, single guide RNAs) combined with the use of the non-integrating AAV6 viral vector to deliver the donor template has been shown to be a highly effective system to modify therapeutically relevant primary human cells including HSPCs, T-cells, and induced pluripotent cells 29 . This approach has shown pre-clinical promise to usher in a new class of medicines for sickle cell disease 27,30 , SCID-X1 31,32 , MPS I 33 , chronic granulomatous disease 34 , X-linked Hyper IgM 35 , and cancer 36 . The specificity of genome editing, however, means that with current approaches it is not possible to track the output of any specific gene modified cell.…”

Advances in the gene therapy of monogenic blood cell diseases

“…HSCs using a CRISPR-Cas9/AAV6-based strategy for the integration of the IL2RG cDNA into the endogenous start codon of this gene. 164 Additional studies confirmed the possibility of editing SCD HSCs by the use of ZFNs combined with IDLVs to deliver the therapeutic cDNA into the β-globin gene. 165 Our group also showed the phenotypic correction of HSPCs from FA patients through the specific integration of FANCA in the AAVS1 locus using a similar combination of ZFN mRNAs and therapeutic donor IDLVs.…”

“…The same study showed the feasibility of correcting BM HSCs from SCID‐X1 patients by means of the insertion of exons 5 to 8 of IL2RG cDNA in the endogenous IL2RG gene. A more recent study has shown gene correction of SCID‐X1 HSCs using a CRISPR‐Cas9/AAV6‐based strategy for the integration of the IL2RG cDNA into the endogenous start codon of this gene . Additional studies confirmed the possibility of editing SCD HSCs by the use of ZFNs combined with IDLVs to deliver the therapeutic cDNA into the β‐globin gene .…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…While most X‐SCID patients treated with integrating retroviral vectors have shown a marked clinical benefit, targeted genome editing with CRISPR/Cas9 may enhance the precision and safety of gene therapy. Pavel‐Dinu et al () have recently described a CRISPR/Cas9‐based method for correcting X‐SCID. This universal strategy, analogous to that previously described for β‐thalassaemia, integrates a codon‐optimized IL2RG cDNA by HR into the endogenous IL2RG start codon.…”

“…Later studies have shown that these Cas9 variants display drastically reduced on‐target activities when delivered as RNP complex (Vakulskas et al , ). A new variant developed using a large high‐throughput assay that screened about 250 000 Cas9 mutants produced the HiFi SpCas9 variant, which only displays a moderate decrease in on‐target efficiencies while vastly reducing off‐target activity (Gomez‐Ospina et al , ; Pavel‐Dinu et al , ; Vakulskas et al , ).…”