Evolving Gene Therapy in Primary Immunodeficiency
Authors: Adrian J. Thrasher, David A. Williams
Journal: Molecular Therapy (2017)

Prior to the first successful bone marrow transplant in 1968, patients born with severe combined immunodeficiency (SCID) invariably died. Today, with a widening availability of newborn screening, major improvements in the application of allogeneic procedures, and the emergence of successful hematopoietic stem and progenitor cell (HSC/P) gene therapy, the majority of these children can be identified and cured. Here, we trace key steps in the development of clinical gene therapy for SCID and other primary immunodeficiencies (PIDs), and review the prospects for adoption of new targets and technologies. Prior to the first successful bone marrow transplant in 1968, patients born with severe combined immunodeficiency (SCID) invariably died. Today, with a widening availability of newborn screening, major improvements in the application of allogeneic procedures, and the emergence of successful hematopoietic stem and progenitor cell (HSC/P) gene therapy, the majority of these children can be identified and cured. Here, we trace key steps in the development of clinical gene therapy for SCID and other primary immunodeficiencies (PIDs), and review the prospects for adoption of new targets and technologies. Primary immunodeficiencies (PIDs) are a diverse group of rare, largely monogenic disorders that result in variable susceptibility to infection, autoimmunity/inflammation, and in some cases, malignancy. More than 300 genes have now been associated with syndromic and non-syndromic PID, including the most frequently occurring forms.1Picard C. Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. Cunningham-Rundles C. Etzioni A. Holland S.M. Klein C. et al.Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015.J. Clin. Immunol. 2015; 35: 696-726Crossref PubMed Scopus (117) Google Scholar The severest PIDs present early in life with failure to thrive and severe infections (often opportunistic), and they are usually fatal unless definitive therapy can be implemented rapidly.2Fischer A. Notarangelo L.D. Neven B. Cavazzana M. Puck J.M. Severe combined immunodeficiencies and related disorders.Nat. Rev. Dis. Primers. 2015; 1: 15061Crossref PubMed Google Scholar Fortunately, many of these immunological defects are intrinsic to the hematopoietic system, making them tractable targets for allogeneic hematopoietic stem cell transplantation (HSCT). Since the first successful bone marrow transplants in two PIDs, X-linked severe combined immunodeficiency (SCID) (SCID-X1) and Wiskott-Aldrich syndrome (WAS), HSCT methodologies and technologies (including conditioning regimens) have improved, and the morbidity and mortality associated with this procedure have diminished considerably.3Gatti R.A. Meuwissen H.J. Allen H.D. Hong R. Good R.A. Immunological reconstitution of sex-linked lymphopenic immunological deficiency.Lancet. 1968; 2: 1366-1369Abstract PubMed Google Scholar, 4Bach F.H. Albertini R.J. Joo P. Anderson J.L. Bortin M.M. Bone-marrow transplantation in a patient with the Wiskott-Aldrich syndrome.Lancet. 1968; 2: 1364-1366Abstract PubMed Google Scholar, 5Gennery A.R. Slatter M.A. Grandin L. Taupin P. Cant A.J. Veys P. Amrolia P.J. Gaspar H.B. Davies E.G. Friedrich W. et al.Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation; European Society for ImmunodeficiencyTransplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better?.J. Allergy Clin. Immunol. 2010; 126 (e1–e11): 602-610Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Booth C. Silva J. Veys P. Stem cell transplantation for the treatment of immunodeficiency in children: current status and hopes for the future.Expert Rev. Clin. Immunol. 2016; 12: 713-723Crossref PubMed Scopus (0) Google Scholar Even so, for patients without HLA-matched donors, graft-versus-host disease (GvHD), delayed immunological reconstitution, and graft rejection remain significant problems. Some conditions are better candidates than others due to disease-specific characteristics, and risks are heightened in patients who are actively infected or who have developed chronic complications at the time of treatment. More than 30 years ago, the demonstration of retroviral gene transfer to hematopoietic stem and progenitor cells (HSC/Ps) led to the suggestion that this technology could provide an alternative platform for development of therapies in a number of diseases that were amenable to HSCT.7Joyner A. Keller G. Phillips R.A. Bernstein A. Retrovirus transfer of a bacterial gene into mouse haematopoietic progenitor cells.Nature. 1983; 305: 556-558Crossref PubMed Google Scholar, 8Williams D.A. Lemischka I.R. Nathan D.G. Mulligan R.C. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse.Nature. 1984; 310: 476-480Crossref PubMed Scopus (263) Google Scholar, 9Dick J.E. Magli M.C. Huszar D. Phillips R.A. Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice.Cell. 1985; 42: 71-79Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 10Keller G. Paige C. Gilboa E. Wagner E.F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors.Nature. 1985; 318: 149-154Crossref PubMed Google Scholar, 11Gruber H.E. Finley K.D. Hershberg R.M. Katzman S.S. Laikind P.K. Seegmiller J.E. Friedmann T. Yee J.K. Jolly D.J. Retroviral vector-mediated gene transfer into human hematopoietic progenitor cells.Science. 1985; 230: 1057-1061Crossref PubMed Scopus (0) Google Scholar The advantages of autologous gene therapy were anticipated to lie in the lack of need to identify a suitable donor, obviation of GvHD, and the potential to reduce the risks of myelosuppressive and immunosuppressive preconditioning of the patient, which is required to make space for engrafting HSC/Ps. For some conditions it was expected that corrected cells would have a profound growth and survival advantage, allowing reconstitution from relatively low numbers of cells. In addition, expression of transgenes at supraphysiological levels was anticipated to allow "cross-correction" of other deficient non-hematopoietic cells in some diseases.12Williams D.A. Broadening the indications for hematopoietic stem cell genetic therapies.Cell Stem Cell. 2013; 13: 263-264Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Over a number of years, successful correction of cellular and animal models boosted expectation that gene therapy would rapidly become mainstream, but clinical translation proved more difficult (Figure 1). This partly related to a limited understanding of human HSC/P culture conditions necessary to achieve high-level gene transfer ex vivo, yet at the same time retaining in vivo engraftment capability, which was not well modeled in murine preclinical transplant assays.13Williams D.A. Principles of cell-based genetic therapies.in: Hoffman R. Benz J. Silberstein L.E. Heslop H. Weitz J. Anastasi J. Hematology: Basic Principles and Practice. Elsevier, 2013: 1503-1512Google Scholar Furthermore, the occurrence of clinically manifesting insertional mutagenesis highlighted deficiencies in early retroviral vector technology that would have to be resolved.14Baum C. von Kalle C. Staal F.J. Li Z. Fehse B. Schmidt M. Weerkamp F. Karlsson S. Wagemaker G. Williams D.A. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.Mol. Ther. 2004; 9: 5-13Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar Adenosine deaminase (ADA) is a housekeeping enzyme of the purine metabolic pathway and is widely expressed.15Hershfield M.S. Adenosine deaminase deficiency: clinical expression, molecular basis, and therapy.Semin. Hematol. 1998; 35: 291-298PubMed Google Scholar The metabolic environment resulting from deficiency of ADA and the accumulation of toxic purine metabolites causes variable skeletal, lung, liver, gastrointestinal, neurodevelopmental, and sensorineural defects, whereas the most consistent and profound abnormality is in the development of lymphocytes, including T, B, and natural killer cells.16Whitmore K.V. Gaspar H.B. Adenosine deaminase deficiency - more than just an immunodeficiency.Front. Immunol. 2016; 7: 314Crossref PubMed Scopus (4) Google Scholar, 17Sauer A.V. Hernandez R.J. Fumagalli F. Bianchi V. Poliani P.L. Dallatomasina C. Riboni E. Politi L.S. Tabucchi A. Carlucci F. et al.Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients.Sci. Rep. 2017; 7: 40136Crossref PubMed Scopus (0) Google Scholar Exogenous polyethylene-glycol-conjugated ADA (PEG-ADA) can rescue the immunological defects through extracellular detoxification, but these effects are often partial and poorly sustained.18Chan B. Wara D. Bastian J. Hershfield M.S. Bohnsack J. Azen C.G. Parkman R. Weinberg K. Kohn D.B. Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID).Clin. Immunol. 2005; 117: 133-143Crossref PubMed Scopus (91) Google Scholar Even so, the use of PEG-ADA at diagnosis has proved a very useful way to stabilize patients before a definitive procedure can be implemented.19Hassan A. Booth C. Brightwell A. Allwood Z. Veys P. Rao K. Hönig M. Friedrich W. Gennery A. Slatter M. et al.Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation and European Society for ImmunodeficiencyOutcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency.Blood. 2012; 120 (quiz 3626): 3615-3624Crossref PubMed Scopus (0) Google Scholar, 20Kohn D.B. Gaspar H.B. How we manage adenosine deaminase-deficient severe combined immune deficiency (ADA SCID).J. Clin. Immunol. 2017; (Published online February 14, 2017)https://doi.org/10.1007/s10875-017-0373-yCrossref PubMed Scopus (2) Google Scholar Several clinical trials were initiated in the early 1990s using gammaretroviral vectors that had been evaluated in murine and primate model systems, and in which ADA cDNA transgene expression was regulated by the retroviral long terminal repeat (LTR). It was also anticipated that the selective growth and survival advantage imparted to gene-corrected cells would obviate the need for preconditioning of the patient. However, these early studies using residual peripheral blood lymphocytes, umbilical cord blood, and bone marrow failed to provide clear clinical benefit over and above that of PEG-ADA, which the patients continued to receive.21Blaese R.M. Culver K.W. Miller A.D. Carter C.S. Fleisher T. Clerici M. Shearer G. Chang L. Chiang Y. Tolstoshev P. et al.T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.Science. 1995; 270: 475-480Crossref PubMed Google Scholar, 22Bordignon C. Notarangelo L.D. Nobili N. Ferrari G. Casorati G. Panina P. Mazzolari E. Maggioni D. Rossi C. Servida P. et al.Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients.Science. 1995; 270: 470-475Crossref PubMed Google Scholar, 23Kohn D.B. Weinberg K.I. Nolta J.A. Heiss L.N. Lenarsky C. Crooks G.M. Hanley M.E. Annett G. Brooks J.S. el-Khoureiy A. et al.Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.Nat. Med. 1995; 1: 1017-1023Crossref PubMed Google Scholar, 24Hoogerbrugge P.M. van Beusechem V.W. Fischer A. Debree M. le Deist F. Perignon J.L. Morgan G. Gaspar B. Fairbanks L.D. Skeoch C.H. et al.Bone marrow gene transfer in three patients with adenosine deaminase deficiency.Gene Ther. 1996; 3: 179-183PubMed Google Scholar Subsequent studies (again using LTR-intact gammaretroviral vectors) introduced some key changes, including the withdrawal of PEG-ADA to enhance the selective advantage of corrected cells, and most importantly the use of low- and/or reduced-intensity conditioning with alkylating agents (usually busulphan, but in a few patients melphalan) to promote engraftment of HSC/Ps.25Aiuti A. Slavin S. Aker M. Ficara F. Deola S. Mortellaro A. Morecki S. Andolfi G. Tabucchi A. Carlucci F. et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (0) Google Scholar, 26Gaspar H.B. Bjorkegren E. Parsley K. Gilmour K.C. King D. Sinclair J. Zhang F. Giannakopoulos A. Adams S. Fairbanks L.D. et al.Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of PEG-ADA and use of mild preconditioning.Mol. Ther. 2006; 14: 505-513Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 27Aiuti A. Cassani B. Andolfi G. Mirolo M. Biasco L. Recchia A. Urbinati F. Valacca C. Scaramuzza S. Aker M. et al.Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.J. Clin. Invest. 2007; 117: 2233-2240Crossref PubMed Scopus (180) Google Scholar, 28Aiuti A. Cattaneo F. Galimberti S. Benninghoff U. Cassani B. Callegaro L. Scaramuzza S. Andolfi G. Mirolo M. Brigida I. et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N. Engl. J. Med. 2009; 360: 447-458Crossref PubMed Scopus (585) Google Scholar, 29Candotti F. Shaw K.L. Muul L. Carbonaro D. Sokolic R. Choi C. Schurman S.H. Garabedian E. Kesserwan C. Jagadeesh G.J. et al.Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.Blood. 2012; 120: 3635-3646Crossref PubMed Scopus (114) Google Scholar, 30Cicalese M.P. Ferrua F. Castagnaro L. Pajno R. Barzaghi F. Giannelli S. Dionisio F. Brigida I. Bonopane M. Casiraghi M. et al.Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency.Blood. 2016; 128: 45-54Crossref PubMed Scopus (0) Google Scholar, 31Shaw K.K. Garabedian E. Mishra S. Barman P. Davila D. Carbonaro D. Shupien S. Silvin C. Geiger S. Nowicki B. et al.Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficiency immunodeficiency.J. Clin. Invest. 2017; https://doi.org/10.1172/JCI90367Crossref Scopus (1) Google Scholar, 32Gaspar H.B. Cooray S. Gilmour K.C. Parsley K.L. Zhang F. Adams S. Bjorkegren E. Bayford J. Brown L. Davies E.G. et al.Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction.Sci. Transl. Med. 2011; 3: 97ra80Crossref PubMed Scopus (117) Google Scholar The combined overall survival rate of around 50 patients treated in these studies was 100%, with a disease-free survival rate (not requiring re-introduction of PEG-ADA or HSCT) of more than 70%. Immunological reconstitution has in general been robust, with high-level gene marking in lymphocytes and, for the first time, albeit at lower levels in myeloid cells, which is an effective surrogate for HSC/P marking in bone marrow. Permanent withdrawal of immunoglobulin supplementation was achieved in approximately 50% of patients, suggesting that further improvements are still possible. Recently, a collaboration among GlaxoSmithKline (GSK), Fondazione Telethon, and Ospedale San Raffaele secured European Marketing Authorisation for Strimvelis TM, which is the original LTR-based vector studied in Milan.22Bordignon C. Notarangelo L.D. Nobili N. Ferrari G. Casorati G. Panina P. Mazzolari E. Maggioni D. Rossi C. Servida P. et al.Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients.Science. 1995; 270: 470-475Crossref PubMed Google Scholar From proof of concept in humans to this first ex vivo market authorization for a gene therapy product, vector technology has advanced considerably. HIV-1-based lentiviruses in particular have been adopted as the current vector of choice for HSC/P gene transfer because they appear to offer some advantages in terms of mutagenicity (because of a more favorable genome insertion profile within transcriptional units rather than at transcription start sites [TSSs], a theoretical advantage), lack of intrinsic retroviral LTR enhancer activity (a modification that has also been duplicated in a new generation of gammaretroviruses), and higher efficiency of gene transfer.33Naldini L. Blömer U. Gallay P. Ory D. Mulligan R. Gage F.H. Verma I.M. Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Google Scholar, 34Naldini L. Trono D. Verma I.M. Lentiviral vectors, two decades later.Science. 2016; 353: 1101-1102Crossref PubMed Scopus (0) Google Scholar, 35Everson E.M. Trobridge G.D. Retroviral vector interactions with hematopoietic cells.Curr. Opin. Virol. 2016; 21: 41-46Crossref PubMed Scopus (0) Google Scholar Following several years of preclinical development, clinical studies using lentiviral vectors are under way for ADA-SCID.36Carbonaro D.A. Zhang L. Jin X. Montiel-Equihua C. Geiger S. Carmo M. Cooper A. Fairbanks L. Kaufman M.L. Sebire N.J. et al.Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.Mol. Ther. 2014; 22: 607-622Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Early indications in more than 32 treated patients, some of whom were identified by newborn screening, are of excellent efficacy and no associated toxicity (H.B. Gaspar and D.B. Kohn, personal communication). Interestingly, no clinically manifesting vector-related toxicities have been observed in any of these studies to date. This is surprising because vectors with similar configurations have caused mutagenesis and malignancy in a number of other conditions, as will be highlighted later.37Li Z. Düllmann J. Schiedlmeier B. Schmidt M. von Kalle C. Meyer J. Forster M. Stocking C. Wahlers A. Frank O. et al.Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (514) Google Scholar, 38Cavazza A. Moiani A. Mavilio F. Mechanisms of retroviral integration and mutagenesis.Hum. Gene Ther. 2013; 24: 119-131Crossref PubMed Scopus (45) Google Scholar Several reports have now detailed a comprehensive analysis of retroviral integration sites (RISs) in cells from ADA-SCID patients treated with LTR-intact gammaretroviral vectors, and although the anticipated patterns of integration are observed, namely preference for TSSs, including sites near the proto-oncogenes Lim domain only 2 (LMO2), BCL2, and CCND2, there are also indications of subtle mutagenic clonal disturbances, although apparently insufficient to drive a frank malignant program (H.B. Gaspar and D.B. Kohn, personal communication).39Cassani B. Montini E. Maruggi G. Ambrosi A. Mirolo M. Selleri S. Biral E. Frugnoli I. Hernandez-Trujillo V. Di Serio C. et al.Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy.Blood. 2009; 114: 3546-3556Crossref PubMed Scopus (48) Google Scholar, 40Biasco L. Ambrosi A. Pellin D. Bartholomae C. Brigida I. Roncarolo M.G. Di Serio C. von Kalle C. Schmidt M. Aiuti A. Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell.EMBO Mol. Med. 2011; 3: 89-101Crossref PubMed Scopus (59) Google Scholar The reason for this disease-specific difference in toxicity is at present unclear, but it could relate to a lower replicative stress in reconstituting T cells on the non-hematopoietic environmental background of ADA deficiency in the thymus, or the presence of competing non-transduced but detoxified thymocytes that successfully complete their maturation program.41Ginn S.L. Hallwirth C.V. Liao S.H.Y. Teber E.T. Arthur J.W. Wu J. Lee H.C. Tay S.S. Reddell R.R. McCormack M.P. et al.Limiting thymic precursor supply increases the risk of lymphoid malignancy in murine X-linked severe combined immunodeficiency.Mol. Ther. 2017; 6: 1-14Google Scholar However, this still does not explain the lack of myeloid toxicity unless a similar argument can be applied to the bone marrow microenvironment or even during ex vivo culture. X-linked SCID (for SCID-X1) is the most common form of SCID, accounting for 40%–50% of all cases.2Fischer A. Notarangelo L.D. Neven B. Cavazzana M. Puck J.M. Severe combined immunodeficiencies and related disorders.Nat. Rev. Dis. Primers. 2015; 1: 15061Crossref PubMed Google Scholar Mutations in the IL2RG gene lead to defective expression of the common cytokine receptor gamma chain (gc), so-called because it is a subunit shared by multiple cytokine receptors, including the interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptor complexes, which are variably involved in the development and function of all lymphocytes. As a consequence, patients show profound defects of adaptive immunity, resulting from the low number or absence of T and natural killer (NK) lymphocytes, and the loss of function of B lymphocytes. The first two clinical trials of gene therapy for SCID-X1 included a total number of 20 subjects and were conducted using virtually identical LTR-intact gammaretroviral vectors and very similar transduction protocols.42Cavazzana-Calvo M. Hacein-Bey S. de Saint Basile G. Gross F. Yvon E. Nusbaum P. Selz F. Hue C. Certain S. Casanova J.L. et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (1861) Google Scholar, 43Gaspar H.B. Parsley K.L. Howe S. King D. Gilmour K.C. Sinclair J. Brouns G. Schmidt M. Von Kalle C. Barington T. et al.Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.Lancet. 2004; 364: 2181-2187Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 44Hacein-Bey-Abina S. Hauer J. Lim A. Picard C. Wang G.P. Berry C.C. Martinache C. Rieux-Laucat F. Latour S. Belohradsky B.H. et al.Efficacy of gene therapy for X-linked severe combined immunodeficiency.N. Engl. J. Med. 2010; 363: 355-364Crossref PubMed Scopus (342) Google Scholar, 45Gaspar H.B. Cooray S. Gilmour K.C. Parsley K.L. Adams S. Howe S.J. Al Ghonaium A. Bayford J. Brown L. Davies E.G. et al.Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency.Sci. Transl. Med. 2011; 3: 97ra79Crossref PubMed Scopus (117) Google Scholar Neither study used preconditioning, with the expectation that the extremely high survival advantage of corrected lymphocyte precursors would allow the development of a highly diverse T cell repertoire (as observed following HLA-matched HSCT infusions, and in some variant patients with rare somatic reversion events).46Stephan V. Wahn V. Le Deist F. Dirksen U. Broker B. Müller-Fleckenstein I. Horneff G. Schroten H. Fischer A. de Saint Basile G. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells.N. Engl. J. Med. 1996; 335: 1563-1567Crossref PubMed Scopus (0) Google Scholar, 47Speckmann C. Pannicke U. Wiech E. Schwarz K. Fisch P. Friedrich W. Niehues T. Gilmour K. Buiting K. Schlesier M. et al.Clinical and immunologic consequences of a somatic reversion in a patient with X-linked severe combined immunodeficiency.Blood. 2008; 112: 4090-4097Crossref PubMed Scopus (27) Google Scholar Successful recovery of a functional T cell compartment occurred in most patients and has been sustained with a disease-free survival rate greater than 80% and excellent overall clinical outcome. In the absence of conditioning, sustained engraftment of hematopoietic stem cells (HSCs) did not occur; consequently, long-term B cell and NK cell marking was very low. Unfortunately, 31–68 months after gene therapy, five patients who were clinically well and had successfully reconstituted developed a T cell acute lymphoblastic leukemia (T-ALL).48Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (2288) Google Scholar, 49Schwarzwaelder K. Howe S.J. Schmidt M. Brugman M.H. Deichmann A. Glimm H. Schmidt S. Prinz C. Wissler M. King D.J. et al.Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.J. Clin. Invest. 2007; 117: 2241-2249Crossref PubMed Scopus (152) Google Scholar, 50Howe S.J. Mansour M.R. Schwarzwaelder K. Bartholomae C. Hubank M. Kempski H. Brugman M.H. Pike-Overzet K. Chatters S.J. de Ridder D. et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J. Clin. Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (615) Google Scholar In four of five patients, enhancer-mediated upregulation of the LMO-2 proto-oncogene was directly implicated in the leukemic process, although an accumulation of more classical genetic changes (many of which have been previously described in de novo T-ALL) unrelated directly to retroviral vector insertion was probably required for final evolution to acute T-ALL.50Howe S.J. Mansour M.R. Schwarzwaelder K. Bartholomae C. Hubank M. Kempski H. Brugman M.H. Pike-Overzet K. Chatters S.J. de Ridder D. et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J. Clin. Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (615) Google Scholar, 51Hacein-Bey-Abina S. Garrigue A. Wang G.P. Soulier J. Lim A. Morillon E. Clappier E. Caccavelli L. Delabesse E. Beldjord K. et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J. Clin. Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (891) Google Scholar It has recently been postulated that intra-thymic replicative stress in the absence of T cell progenitor import from the bone marrow is a significant contributor to the establishment of a leukemic program because HSC engraftment is essentially absent.41Ginn S.L. Hallwirth C.V. Liao S.H.Y. Teber E.T. Arthur J.W. Wu J. Lee H.C. Tay S.S. Reddell R.R. McCormack M.P. et al.Limiting thymic precursor supply increases the risk of lymphoid malignancy in murine X-linked severe combined immunodeficiency.Mol. Ther. 2017; 6: 1-14Google Scholar, 52Martins V.C. Ruggiero E. Schlenner S.M. Madan V. Schmidt M. Fink P.J. von Kalle C. Rodewald H.R. Thymus-autonomous T cell development in the absence of progenitor import.J. Exp. Med. 2012; 209: 1409-1417Crossref PubMed Scopus (45) Google Scholar If this is the case, then use of conditioning would likely reduce risk for clinically manifesting mutagenesis. No convincing evidence could be confirmed for a pathogenic role of non-physiological expression of the transgene in leukemogenesis through aberrant receptor signaling, although, of course, it allowed the T cell maturation to proceed.53Scobie L. Hector R.D. Grant L. Bell M. Nielsen A.A. Meikle S. Philbey A. Thrasher A.J. Cameron E.R. Blyth K. Neil J.C. A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but no evidence of gammaC gene oncogenicity.Mol. Ther. 2009; 17: 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar All patients were treated with relatively standard chemotherapy, but one patient died of refractory leukemia. Remarkably, the four surviving patients recovered their T cell immunity after completion of chemotherapy without need for any further intervention. This suggests that very long-lasting (possibly self-renewing), gene-corrected T cell precursors engrafted in the thymus soon after infusion of cells, and that these are capable of sustaining active thymopoiesis for many years. Molecular studies in mice engrafted with cells transduced with replication-incompetent gammaretroviral vectors identified duplicated enhancer elements in the LTR as the major culprit driving dysregulated gene expression in the neighborhood of the RIS.37Li Z. Düllmann J. Schiedlmeier B. Schmidt M. von Kalle C. Meyer J. Forster M. Stocking C. Wahlers A. Frank O. et al.Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (514) Google Scholar, 54Modlich U. Bohne J. Schmidt M. von Kalle C. Knöss S. Schambach A. Baum C. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (208) Google Scholar, 55Kustikova O. Fehse B. Modlich U. Yang M. Düllmann J. Kamino K. von Neuhoff N. Schlegelberger B. Li Z. Baum C. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (269) Google Scholar This effect was also shown to have activity over quite long ranges and to operate in combination with other molecular lesions.50Howe S.J. Mansour M.R. Schwarzwaelder K. Bartholomae C. Hubank M. Kempski H. Brugman M.H. Pike-Overzet K. Chatters S.J. de Ridder D. et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J. Clin. Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (615) Google Scholar, 51Hacein-Bey-Abina S. Garrigue A. Wang G.P. Soulier J. Lim A. Morillon E. Clappier E. Caccavelli L. Delabesse E. Be