ORIGINAL ARTICLE Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer K Pike-Overzet 1,2 , M Rodijk 1,2 , Y-Y Ng 1,2 , MRM Baert 1,2 , C Lagresle-Peyrou 3,4 , A Schambach 5 , F Zhang 6 , RC Hoeben 7 , S Hacein-Bey-Abina 3,4 , AC Lankester 8 , RGM Bredius 8 , GJA Driessen 9 , AJ Thrasher 6 , C Baum 5 , M Cavazzana-Calvo 3,4 , JJM van Dongen 2 and FJT Staal 1,2 1 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands; 2 Department of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands; 3 Institut National de la Sante ´ et de la Recherche Me ´dicale (INSERM), Paris, France; 4 Department of Biotherapy, Centre d’Investigation Clinique inte ´gre ´ en Biothe ´rapies, Ho ˆ pital Necker-Enfants Malades, Assistance Publique-Ho ˆ pitaux de Paris, Paris, France; 5 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 6 Molecular Immunology Unit, Institute of Child Health, University College London, London, UK; 7 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands; 8 Department of Pediatric Immunology, Hemato-Oncology, Bone Marrow Transplantation and Auto-immune Diseases, Leiden University Medical Center, Leiden, The Netherlands and 9 Department of Pediatrics, Erasmus MC University Medical Center, Rotterdam, The Netherlands 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 Vb gene segment usage was comparable to wild- type (WT) controls, indicating that RAG-mediated rearrange- ment 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. Leukemia (2011) 25, 1471–1483; doi:10.1038/leu.2011.106; published online 27 May 2011 Keywords: gene therapy; T lymphocyte; SCID Introduction Severe combined immunodeficiency (SCID) comprises a group of rare diseases in which lymphocytes are unable to develop or function normally. The phenotype of SCID depends on the underlying genetic defect, but all SCID patients lack autologous T lymphocytes in their peripheral blood. 1–5 SCID patients with a mutation in the recombination activation gene 1 or 2 (RAG1 or RAG2) lack both T and B lymphocytes because RAG proteins are crucial for the rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes. In the absence of these rearrange- ments, functional Ig and TCR chains will not be produced by B- and T-cell progenitors in bone marrow (BM) and thymus. This leads to a developmental arrest causing the T-B-SCID pheno- type. 6–9 Allogeneic hematopoietic stem cell transplantation is a curative treatment for SCID, with the 3-year survival of a human leukocyte antigen-identical transplantation being significantly better than a, more widely available, mismatched transplantation. 10 However, an human leukocyte antigen- identical donor is often not available, so SCID patients mostly have to rely on mismatched donors. Over the past decades, gene therapy utilizing long terminal repeat (LTR)-driven g-retrovirus- based vectors has emerged as an alternative treatment option for SCID. Several clinical trials have been successful in the treatment of adenosine deaminase-SCID and X-linked SCID patients. 11–13 In addition, therapeutic LTR-driven g-retroviral transfer vectors have been developed and tested for other SCID-causing genetic aberrations, such as Artemis, IL7RA, RAG1 and RAG2. 14–17 Unfortunately, clonal T-cell prolifera- tions were observed in 4 out of 10 patients enrolled in the French X-linked SCID gene therapy trial 18,19 and in 1 out of 10 patients in the British X-linked SCID trial. The occurrence of clonal T-cell proliferations has lead to widespread efforts to improve the safety of gene transfer methods. Using a different vector system is one of the options to improve safety (reviewed in Staal et al. 20 ). Over the past 15 years, lentiviral transfer systems underwent several rounds of modifications to improve their safety. 21–24 An additional modification resulted in the lentiviral self-inactivating (SIN) vector system. SIN vectors, both g-retroviral and lentiviral, contain a LTR from which viral promoter–enhancer sequences have been deleted from the U3 region of the 3 0 LTR, leaving the LTR transcriptionally inactive when integrated into the host genome. 22,25,26 In vitro studies have shown that, compared with g-retroviral vectors, lentiviral vectors have a more favorable integration pattern. 27 Integrations of lentiviral vectors are spread out over the transcriptional part of genes, whereas g-retroviral vectors tend to integrate more near DNAse I-hypersensitive sites 28 and toward the transcription start site of genes, 27 regions that mostly contain regulatory elements. Some studies have already shown partial or full phenotypic correction of primary immuno- deficiencies using lentiviral SIN vectors 29,30 indicating that these vectors are a good option for the use in human RAG1 gene transfer. Received 23 December 2010; revised 28 March 2011; accepted 1 April 2011; published online 27 May 2011 Correspondence: Professor Dr FJT Staal, Department of Immunohematol- ogy and Blood Transfusion (IHB), Leiden University Medical Center, PO Box 9600, Albinusdreef 2, IHB-L1-36, 2300 RC Leiden, The Netherlands. E-mail: f.j.t.staal@lumc.nl Leukemia (2011) 25, 1471–1483 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu