Abstract
Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1−/− mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vβ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1−/− mice using lentiviral SIN vectors.
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Acknowledgements
We thank Edwin de Haas for cell sorting and all other members of the Staal laboratory for their technical contributions. We thank Alexandre G Troullioud Lucas for his contribution to testing the coRAG1 vectors. We are grateful for the biotechnical assistance of Yvette Caljouw in the mouse facility. We are grateful to L Naldini (San Raffaele Telethon Institute for Gene Therapy Milano, Italy) for providing the helper plasmids pMDLg/pRRE, pRSV-Rev and pMD2.VSVG, and the original transfer vector. This work was in part supported by grants of the European Union: INHERINET (QLK3-CT-2001-0427) and CONSERT (LSHB-CT-2004-005242), in part by grants of the Netherlands Organization for Health Research and Development (Translational Gene Therapy Research Programme of ZonMw, 43100016 and Innovational Research Incentives Scheme Veni grant, 91610083), Kika (Children Cancer Free, 09/036) and REBIRTH Excellence Cluster (Exc62/1).
Author contributions
KPO, FJTS, JJMvD and CB designed research; KPO, MR, YYN and MRMB performed experiments; AS, FZ, AJT and CB contributed vital new reagents; KPO, MR and FJTS analyzed data; KPO and FJTS interpreted data; YYN, AS, RCH, ACL, RGMB, GJAD, AJT, CB, MCC, JJMvD, AL and RH contributed in essential discussions. CL, SHBA and MCC contributed unpublished experiments that helped the direction of the work; KPO and FJTS wrote manuscript.
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Pike-Overzet, K., Rodijk, M., Ng, YY. et al. Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer. Leukemia 25, 1471–1483 (2011). https://doi.org/10.1038/leu.2011.106
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DOI: https://doi.org/10.1038/leu.2011.106
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