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

Schiroli G, Ferrari S, Conway A, Jacob A, Capo V, Albano L, Plati T, Castiello MC, Sanvito F, Gennery AR, Bovolenta C, Palchaudhuri R, Scadden DT, Holmes MC, Villa A, Sitia G, Lombardo A, Genovese P, Naldini L
Source: Science
Publication Date: (2017)
Issue: 9(411): eaan0820
Research Area:
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector® LV-Unit

Primary T lymphocytes from healthy donors’ PB mononuclear cells were isolated and activated using magnetic beads conjugated to anti-human CD3 and CD28 antibodies (Dynabeads human T-activator CD3/CD28; Invitrogen) in IMDM medium (GIBCO-BRL) supplemented with penicillin, streptomycin, glutamine, 10% FBS, and 5 ng/ml of IL-7 and IL-15 (PeproTech) as described (42). After 2 days of stimulation, T cells were infected with the indicated IDLVs at MOI of 100. The following day, transduced T cells were electroporated with mRNAs encoding for ZFNs (106 cells/condition, 100 µg/ml of ZFNs; P3 Primary Cell 4D-Nucleofector X Kit, program EO 115; Lonza) and then expanded to perform flow cytometry and molecular analyses.

For CRISPR/Cas9 editing in human CD34+ cells (from Lonza), 2.5 µM of ribonucleoproteins (RNP) were electroporated. RNPs were made by incubating Cas9 protein (Integrated DNA Technologies) with synthetic 2’O-methyl-phosphorotyoate modified (10) sgRNA (Metabion, HPLC purified, intron 1 IL2R Gguide 8) at 1:1.5 molar ratio for 10 min at 25°C. For large scale experiments, electroporation was performed using Lonza 4D-Nucleofector LV Unit (P3 primary cell nucleofectorkit, program EO 100, Lonza).


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 IL2RG (interleukin-2 receptor common ?-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 IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG 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.