Challenges in Vector and Trial Design Using Retroviral Vectors for Long-Term Gene Correction in Hematopoietic Stem Cell Gene Therapy
Authors: Jacqueline Corrigan-Curay, Odile Cohen-Haguenauer, Marina O'Reilly, Susan R. Ross, Hung Fan, Naomi Rosenberg, Nikunj Somia, Nancy King, Theodore Friedmann, Cynthia Dunbar, Alessandro Aiuti, Luigi Naldini, Christopher Baum, Christof von Kalle, Hans-Peter Kiem, Eugenio Montini, Frederic Bushman, Brian P Sorrentino, Manuel Carrondo, Harry Malech, Gösta Gahrton, Robyn Shapiro, Linda Wolff, Eugene Rosenthal, Robert Jambou, John Zaia, Donald B Kohn
Journal: Molecular Therapy (2012)

Over the past two decades, incredible progress has been made using gene therapy for inherited severe immunodeficiency disorders, such as X-linked severe combined immunodeficiency disorder (SCID-X1) and adenosine deaminase deficiency–severe combined immunodeficiency disorder (ADA-SCID).1Hacein-Bey-Abina S Hauer J Lim A Picard C Wang GP Berry CC et al.Efficacy of gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2010; 363: 355-364Crossref PubMed Scopus (499) Google Scholar,2Booth C Gaspar HB Thrasher AJ Gene therapy for primary immunodeficiency.Curr Opin Pediatr. 2011; 23: 659-666Crossref PubMed Scopus (25) Google Scholar,3Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (816) Google Scholar However, for reasons that remain unclear, gene transfer for SCID-X1 has also been associated with some cases of vector-induced leukemia whereas no cases have been seen in the ADA-SCID trials despite the common use of g-retroviral vectors. The first case was reported in a French gene transfer trial for SCID-X1.4Hacein-Bey-Abina S von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (3001) Google Scholar Over the next six years, an additional three cases were reported in that trial and one in a second SCID-X1 trial that enrolled a combined total of 20 subjects.2Booth C Gaspar HB Thrasher AJ Gene therapy for primary immunodeficiency.Curr Opin Pediatr. 2011; 23: 659-666Crossref PubMed Scopus (25) Google Scholar Unfortunately, genotoxicity would not remain confined to SCID-X1. Recent reports of insertional mutagenesis leading to myelodysplastic syndrome in a trial for chronic granulomatous disease and a case of leukemia in a trial for Wiskott-Aldrich syndrome (WAS), both of which used g-retroviral vectors, underscored that this type of toxicity can also apply to other disease settings.5Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (983) Google Scholar,6Persons DA Baum C Solving the problem of g-retroviral vectors containing long terminal repeats.Mol Ther. 2011; 19: 229-231Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar,7Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (631) Google Scholar In all these cases, insertion of the g-retroviral vector near known proto-oncogenes led to enhancer-mediated expression of these proto-oncogenes. To address this toxicity, investigators have developed new vectors that, in experimental models, can achieve long-term gene correction with less risk of insertional mutagenesis. The field is also seeking to develop new animal models and in vitro assays that can accurately predict whether these vectors will indeed reduce the risk of insertional mutagenesis. In light of these innovations and new trials, the National Institutes of Health (NIH) Office of Biotechnology Activities, in partnership with the NIH Recombinant DNA Advisory Committee and the European Network for the Advancement of Clinical Gene Transfer and Therapy (CliniGene; http://www.clinigene.eu) hosted a conference in December 2010 to examine new developments in this field so as to inform the scientific community, regulators, and the public. The focus of the conference was not only on the scientific advances but also on the potential development of predictive assays, regulatory oversight challenges, and ethical questions regarding the design of new trials with these vectors. This article reviews the key issues raised during that conference. (The presentations and the webcasts for the conference are available at http://oba.od.nih.gov/rdna/rdna_symposia.html#CONF_003h.) Some of the earliest trials using g-retroviral vectors for long-term gene correction focused on the severe combined immunodeficiency disorders including ADA-SCID and SCID-X1 and on the neutrophil oxidase–deficiency chronic granulomatous disease (Table 1).Table 1Clinical experience with vectorsSpeakerDisorderVector typeConditioningResultIOOther outcomesMalechSCID-X1 (infants)γ -RVNoneMost with normal T-, low-normal B-, and NK-cell reconstitution5/20 T cell LPD LMO217 subjects with good growth and development; 2 deaths1Hacein-Bey-Abina S Hauer J Lim A Picard C Wang GP Berry CC et al.Efficacy of gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2010; 363: 355-364Crossref PubMed Scopus (499) Google Scholar, 21Artus A Duros C Botbol Y Scholtz S Schmidt M von Kalle C et al.Directed integration of insulated lentiviral vectors to the heterochromatin towards safer gene transfer to stem cell (abstract 389).Presented 20 May 2011 at the Annual Meeting of the Society of Gene and Cell Therapy, Seattle, WA. 2011Google Scholar, 52Gaspar HB Cooray S Gilmour KC Parsley KL Adams S Howe SJ 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 (98) Google ScholarSCID-X1 (children)γ -RVNoneMinimal engraftment gene- marked cells0/5Minimal; immune improvementSCID-X1 (infants)SIN γ -RV SIN LVNoneOpening 2011-2012−−Chronic granulomatous diseaseγ -RVNoneLow frequency, transient oxidase (+) neutrophils for a few months0Ref. 53Kang E Choi U Theobald N Linton G Long Priel D Kuhns D et al.Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils.Blood. 2010; 115: 783-791Crossref PubMed Scopus (149) Google ScholarChronic granulomatous diseaseγ -RVBusulfan (medium)>10% oxidase-positive neutrophils2/2 MDS EVILInfections cleared; one death6Persons DA Baum C Solving the problem of g-retroviral vectors containing long terminal repeats.Mol Ther. 2011; 19: 229-231Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 8Cartier N Hacein-Bey-Abina S Bartholomae CC Veres G Schmidt M Kutschera I et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1177) Google ScholarAiutiADA-SCID (Milan)γ -RVBusulfan (low)Multilineage marking (0. 1-10% myeloid cells) polyclonal T-cell reconstitution, low-normal B- and NK-cell reconstitution; ADA metabolic detoxification0/1715 subjects off PEGADA, good growth and development; no deaths; 3 delayed neutrophils recovery3Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (816) Google Scholar (A. Aiuti, unpublished data)ADA-SCID (London)γ -RVMelphalan (low) or busulfan (low)Multilineage marking (0. 1-10% myeloid cells) polyclonal T-cell reconstitution, low-normal B- and NK-cell reconstitution; ADA metabolic detoxification0/64 subjects off PEGADA; good growth and development54Gaspar HB Cooray S Gilmour KC Parsley KL Zhang F Adams S 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 (242) Google ScholarADA-SCID Los Angeles/Bethesdaγ -RV4 with no conditioning 14, busulfan (low)Multilineage marking polyclonal T-cell reconstitution, low-normal B- and NK-cell reconstitution; ADA metabolic detoxification0/184 subjects no conditioning: minimal gene marking; 3/6 with busulfan, off PEGADA (F. Candotti, unpublished data)Wiskott-Aldrich syndromeLVBusulfan (medium); fludarabineStable gene marking over first 6 months0/1Treated recentlyNaldiniMetachromatic leukodystrophyLVBusulfan (full)Stable gene marking over first 6 months0/1Treated recentlyvon KalleWiskott-Aldrich syndromeγ -RVBusulfan (medium)Immune reconstitution, platelet recovery1/10 T cell LPD LMO2Ref. 55Boztug K Schmidt M Schwarzer A Banerjee PP Díez IA Dewey RA et al.Stem-cell gene therapy for the Wiskott-Aldrich syndrome.N Engl J Med. 2010; 363: 1918-1927Crossref PubMed Scopus (435) Google ScholarX-adrenoleukodystrophyLVBusulfan (full); cyclophosphamideNeurological stabilization; polyclonal gene marking0/2Ref. 8Cartier N Hacein-Bey-Abina S Bartholomae CC Veres G Schmidt M Kutschera I et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1177) Google ScholarLeboulchβ-thalassemiaLVBusulfan (full)Transfusion independent; clonal dominance½ HMG2ARef. 10Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google ScholarADA-SCID, adenosine deaminase deficiency-severe combined immunodeficiency disorder; IO, insertional oncogenesis, frequency, affected lineage, implicated trans-activated cell gene; LPD, lymphoproliferative disorder; MDS, myelodysplastic syndrome; PEG-ADA, pegademase bovine; SCID-XI, X-linked severe combined immunodeficiency disorder. Open table in a new tab ADA-SCID, adenosine deaminase deficiency-severe combined immunodeficiency disorder; IO, insertional oncogenesis, frequency, affected lineage, implicated trans-activated cell gene; LPD, lymphoproliferative disorder; MDS, myelodysplastic syndrome; PEG-ADA, pegademase bovine; SCID-XI, X-linked severe combined immunodeficiency disorder. Review of these g-retroviral clinical trials demonstrated that gene transfer has the potential to provide long-term gene correction that is comparable to alternative therapies, such as bone marrow transplant (BMT), and—depending on the disease, the age of the patient at diagnosis, and the availability of alternative therapy—gene transfer may prove equally efficacious with potentially less toxicity. However, the factors leading to development of genotoxicity from integration of the vector in a particular trial or subject are not completely understood. Early data on trials using lentiviral vectors were also discussed. Lentiviral vectors are being used in trials for leukodystrophies, including adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), and WAS. In MLD, a defect in the enzyme arylsulfatase A leads to impaired development of the myelin sheath that insulates nerve fibers. ALD, an X-linked disorder, is caused by a mutation in the ALD protein resulting in disruption of myelin and early death due to demyelination. In 2009, in a trial administering autologous CD34+ hematopoietic cells transduced with a lentiviral vector expressing the ALD protein, two subjects achieved polyclonal reconstitution with 9–14% of granulocytes, monocytes, and T and B lymphocytes containing the transferred ALDP gene and that, beginning at 14–16 months, progressive demyelination stopped.8Cartier N Hacein-Bey-Abina S Bartholomae CC Veres G Schmidt M Kutschera I et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1177) Google Scholar Luigi Naldini reported the results of the first treated patient in the MLD trial who received autologous CD34+ cells transduced with a self-inactivating (SIN) lentiviral vector containing arylsulfatase A complementary DNA following busulfan preconditioning. At 3- to 6-month follow-ups, there was sustained gene marking, and expression of the arylsulfatase enzyme at levels 5- to 10-fold above normal were detected in the peripheral blood cells. Eugenio Montini, via analysis of unique integration sites in that subject, demonstrated polyclonal hematopoiesis and displayed a genomic integration profile similar to that of ALD patients and in human mouse hematochimeras.9Biffi A Bartolomae C Cesana D Cartier N Aubourg P Ranzani M et al.Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection.Blood. 2011; 117: 5332-5339Crossref PubMed Scopus (181) Google Scholar Alessandro Aiuti presented the preclinical data and rationale supporting a novel clinical trial for WAS using a lentiviral vector encoding WAS protein under the control of its homologous promoter. A clinical trial using lentiviral vector-transduced autologous CD34+ cells combined with reduced-intensity conditioning has started at his institution, and the first subject was treated in June 2010. Whereas both of these trials reported polyclonal hematopoiesis, another trial, using a lentiviral vector for b-thalassemia, reported the development of an oligoclonal population of vector-containing cells. An HIV-based SIN vector expressing a functional b-globin gene was introduced into hematopoietic stem cells with the goal of correcting deficiencies in hemoglobin production, the underlying defect in b-thalassemia. The first study subject engrafted with the transduced cells has shown improved hemoglobin production and displayed significant clinical improvement.10Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google Scholar However, a clone carrying the gene transfer vector has become dominant. The clone overexpresses the gene encoding HMGA2, a protein that regulates gene transcription. Analyses reveal that cells bearing this insertion express a truncated form of HMGA2 messenger RNA (mRNA) lacking 3′ binding sites for a regulatory microRNA (miRNA) that arises as a consequence of aberrant splicing to a cryptic splice acceptor site in the vector insulator sequences. In contrast to findings in the earlier g-retrovirus vector-based SCID-X1 trials, in which five study subjects developed cases of leukemia associated with vector-mediated activation of LMO2, the subject in the b-thalassemia trial has remained healthy for nearly four years. In addition, there is no evidence of other abnormalities, including karyotype changes, abnormal blood counts, or cytology. Aberrant expression of HMGA2 has been associated with lipomas and some hematological disorders including malignancies.11Fusco A Fedele M Roles of HMGA proteins in cancer.Nat Rev Cancer. 2007; 7: 899-910Crossref PubMed Scopus (574) Google Scholar,12Inoue N Izui-Sarumaru T Murakami Y Endo Y Nishimura J Kurokawa K et al.Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH).Blood. 2006; 108: 4232-4236Crossref PubMed Scopus (118) Google Scholar However, integrations similar to the one discussed here have also been detected in the SCID-X1 trials in study subjects who remain healthy.13Wang GP Berry CC Malani N Leboulch P Fischer A Hacein-Bey-Abina S et al.Dynamics of gene-modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID-X1 gene therapy trial.Blood. 2010; 115: 4356-4366Crossref PubMed Scopus (102) Google Scholar The mechanism(s) that led to partial clonal dominance of this clone are not fully understood. A stochastic event related to low transduction of hematopoietic stem cells could be responsible. Alternatively, the integration affecting HMGA2 may confer a homeostatic advantage to cells. A recent article demonstrated that HMGA2 overexpression could result in benign hemato­poietic stem and progenitor cell expansion in a murine model.14Ikeda K Mason P Bessler M 3′UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis by conferring a clonal growth advantage at the level of HSC in mice.Blood. 2011; 117: 5860-5869Crossref PubMed Scopus (78) Google Scholar Although the clonal population of cells detected in the b-thalassemia trial has remained clinically benign, it is illustrative of how vector insertion can affect adjacent cellular gene expression through non-enhancer-mediated mechanisms. There are essentially four "alternative" mechanisms other than oncogenic activation by which retroviruses and, more recently, transposons have been shown to induce cancer in animal models: alternative splicing, gene inactivation, truncation of cellular mRNA or protein, and miRNA activation. Drawing from in vitro and in vivo preclinical models, numerous examples were presented, in which each of the four alternative mechanisms was responsible for transformation by both replication-competent retroviruses and translocating Sleeping Beauty (SB) transposons (Table 2). In contrast to murine leukemia viruses (MLVs), which primarily cause hemato­poietic lineage cancers, genetically engineered SB transposons have been used to induce tumors in many different cell types. Linda Wolff presented several murine models in which infection with replication-competent MLV causes a variety of hematopoietic cancers, including erythro­leukemias, myeloid tumors, and T- and B-cell lymphomas/leukemias, by non-enhancer-mediated mechanisms. David Largaespada, using the SB system, provided evidence that alternative mechanisms of oncogenesis are not limited to hematopoietic tumors.Table 2Non-enhancer-mediated mechanisms by which integrating elements affect expression of adjacent cellular genesIntegrating elementGeneOutcomeMechanismRef.MLVC-MYB, Notch-1Leukemia, lymphomaProduce truncated mRNA; protein with increased stability, activity56Bies J Feikova S Bottaro D Wolff L Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/Thr protein phosphatases.Oncogene. 2000; 19: 2846-2854Crossref PubMed Scopus (26) Google Scholar, 57Bies J Wolff L Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway.Oncogene. 1997; 14: 203-212Crossref PubMed Scopus (59) Google Scholar, 58Hoemann C Beaulieu N Girard L Rebai N Jolicoeur P Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice.Mol Cell Biol. 2000; 20: 3831-3842Crossref PubMed Scopus (79) Google ScholarSBBRAF, intron 9FibrosarcomaChimeric transcript produces kinase-only BRAF acting as dominant oncogene59Collier LS Carlson CM Ravimohan S Dupuy AJ Largaespada DA Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse.Nature. 2005; 436: 272-276Crossref PubMed Scopus (348) Google ScholarSBEGFR C-terminal exonHepatocellular carcinomaLoss of C-terminal regulatory domain of EGFR, resulting in constitutively active receptor60Keng V Villanueva A Chiang DY Dupuy AJ Ryan BJ Matise I et al.A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.Nat Biotechnol. 2009; 27: 264-274Crossref PubMed Scopus (169) Google ScholarMLVGFI1 3' untranslated regionLymphomaLoss of binding sites for miRNA that negatively regulates Gfi1, increasing expression level61Dabrowska MJ Dybkaer K Johnsen HE Wang B Wabl M Pedersen FS Loss of microRNA targets in the 3′ untranslated region as a mechanism of retroviral insertional activation of growth factor independence 1.J Virol. 2009; 83: 8051-8061Crossref PubMed Scopus (24) Google Scholaranalogous to clinical dominance with lentiviral vector integration into the HMGA2 gene in a β-thalassemia subject10Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google ScholarFriend MLV, Friend SFFVP53ErythroleukemiaInsertional inactivation of disrupted tumor suppressor gene62Ben David Y Prideaux VR Chow V Benchimol S Bernstein A Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.Oncogene. 1988; 3: 179-185PubMed Google ScholarMLVNF1Myeloid leukemia63Cho BC Shaughnessy Jr, JD Largaespada DA Bedigian HG Buchberg AM Jenkins NA et al.Frequent disruption of the Nf1 gene by a novel murine AIDS virus-related provirus in BXH-2 murine myeloid lymphomas.J Virol. 1995; 69: 7138-7146PubMed Google ScholarMLV in p19ARF and p53-deficient miceVariety of putative tumor suppressorsLymphoma64Uren AG Kool J Matentzoglu K de Ridder J Mattison J van Uitert M et al.Large-scale mutagenesis in p19ARF- and p53-deficient mice identifies cancer genes and their collaborative networks.Cell. 2008; 133: 727-741Abstract Full Text Full Text PDF PubMed Scopus (152) Google ScholarSBNFI1, PTENPeripheral nerve sheath tumorsDisruption of tumor suppressor geneD. Largaespada (unpublished data)SBWACColorectal carcinomaMLVIKAROS—intron between exons 2 and 3Lymphoma/acute lymphoblastic leukemiaExpression of truncated dominant negative isoforms65Beverly LJ Capobianco AJ Perturbation of Ikaros isoform selection by MLV integration is a cooperative event in NotchIC-induced T cell leukemogenesis.Cancer Cell. 2003; 3: 551-564Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 66Dail M Li Q McDaniel A Wong J Akagi K Huang B et al.Mutant Ikzf1, KrasG12D, and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents.Proc Natl Acad Sci USA. 2010; 107: 5106-5111Crossref PubMed Scopus (55) Google Scholar Open table in a new tab A common theme gathered from research on retroviruses, transposon systems such as SB, and oncogenesis is that it may take multiple hits to different cellular genes (genetic or epigenetic) to convert a normal cell into a tumor cell. Many mice transgenic for a single proto-oncogene have long latency periods for the development of tumors, and crossing two lines carrying "cooperating" oncogenes (e.g., c-myc and Runx2) can result in more rapid tumor development.15Vaillant F Blyth K Terry A Bell M Cameron ER Neil J et al.A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with MYC.Oncogene. 1999; 18: 7124-7134Crossref PubMed Scopus (80) Google Scholar Because of the requirement for multiple events, many models, including those discussed by Wolff and Largaespada, incorporate the use of mice with genetic predisposition to particular cancers (e.g., tumor suppressor gene knockouts or transgenics that express different oncogenes), chemical mutagens, or inflammatory agents (e.g., pristane) to accelerate transformation by the integrating elements (retroviruses/transposons). However, malignancies secondary to retrovirus-induced changes still occur in wild-type animals, albeit with lower incidence and longer latency. Oncogenesis by retroviral gene therapy vectors may also be influenced by age and genetics. Marc Sitbon reviewed MLV leukemogenesis and pointed out that both age and genetic background influence the efficiency and specificity of disease. In humans, individual genetic makeup may also influence the likelihood of developing a tumor after retroviral gene therapy. MLV infection of young (neonatal) animals leads to much more efficient development of leukemias than infection with the same virus in adults. This could reflect the increased availability of target cells with the potential to become leukemic in young animals or to more robust antiviral immunity in older animals. These findings in mice suggest that human gene transfer by retroviral vectors in neonates or young children may entail a higher oncogenic risk than in older subjects. However, younger age may also underlie the better clinical responses that have been seen for gene transfer to hematopoietic stem cells (HSCs) with integrating vectors, such as the infants with SCID. Lee Ratner discussed human T-lymphotrophic virus type 1, which has direct transforming activity and causes adult T-cell leukemia in humans,16Kannian P Green PL Human T lymphotropic virus type 1 (HTLV-1): molecular biology and oncogenesis.Viruses. 2010; 2: 2037-2077Crossref PubMed Scopus (95) Google Scholar and the long latency between infection and oncogenesis. Interestingly, almost all infections that result in leukemia occur early in life through vertical transmission (predominantly breastfeeding),17Take H Umemoto M Kusuhara K Kuraya K Transmission routes of HTLV-I: an analysis of 66 families.Jpn J Cancer Res. 1993; 84: 1265-1267Crossref PubMed Scopus (28) Google Scholar but the time between infection and development of leukemia is typically several decades, and only about 5% of infected individuals ultimately develop adult T-cell leukemia. By contrast, infection of adults is associated with a neuro­logical disease that occurs with a shorter latency. Of relevance to the field of gene transfer is that even when a retrovirus carries genes with transforming potential, the lag between infection and development of a tumor can be decades, again underscoring the importance of long-term follow-up of subjects in gene correction trials. If activation or inactivation of a single cellular gene does not immediately result in development of oncogenesis, tumori­genicity may take years to develop, leading to a false presumption of safety. Altogether, these observations of the cooperative effects of multiple integrations into different genes have implications for considering oncogenic risk from integrating vectors in gene transfer trials. On the positive side, a single genetic hit (e.g., insertional activation or mutagenesis of one cellular gene) may not be sufficient to induce a tumor cell by itself. Minimizing the numbers of vector integrants per target cell while achieving sufficient percentages of gene-modified cells should be a goal in clinical applications. On the negative side, it has long been recognized that human cancers are the result of multiple genetic changes; dysregulation of cellular gene expression by vector insertion could be an initiating event, and long-term follow-up of subjects may be required. Following the reports of severe adverse events directly attributable to gene transfer vectors integration, several approaches have been pursued separately or in combination, as follows: (i) genetic insulator elements (GIEs) to act as both enhancer-blockers and boundary against potential silencing; (ii) inhibition of integration by knocking down in target cells lens epithelium-derived growth factor (LEDGF), the cell partner of HIV integrase, which may also have the potential to allow integration to be redirected toward defined genetic regions; (iii) selection of genetic regions as safe harbors for integration; (iv) alternative integration systems with a more random integration profile in human cells, such as foamy virus vectors or nonviral vectors derived from transposons; and (v) inhibition of unwanted expression of the transgene in nontarget cells. Odile Cohen-Haguenauer reviewed studies of safety modifications in MLV and lentiviral vectors to determine whether new short synthetic transcriptional insulator modules, identified in collaboration with Nic Mermod (University of Lausanne), could inhibit cis-acting transcriptional effects.18Gaussin A Modlich U Bauche C Niederländer NJ Schambach A Duros C et al.CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.Gene Ther. 2012; 19: 15-24Crossref PubMed Scopus (27) Google Scholar Further studies of a particular GIE that showed sustained and robust expression were undertaken to establish how the system would perform in primary cells. Of note, in a study with David Klatzmann (Paris, France), a strong CD4-specific promoter was constrained by the insulators in human kidney 293 cells, and expression in Jurkat T-cell lines was observed. Comparative high-throughput integration-site analysis performed with Christof von Kalle in collaboration with Montini, using an in vivo genotoxicity assay on Cdkn2a−/− tumor-prone mice,19Montini E Cesana D Schmidt M Sanvito F Bartholomae CC Ranzani M et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (462) Google Scholar provided evidence that this new GIE system might lead to a lower risk of genotoxicity.20Duros C Artus A Gaussin A Scholtz S Cesana D Montini E et al.Insulated lentiviral vectors towards safer gene transfer to stem cells (abstract 383).Presented 20 May 2011 at the Annual Meeting of the Society of Gene and Cell Therapy, Seattle, WA. 2011Google Scholar Insulated lentiviral vectors expressing the Fanconi anemia A complementary DNA are currently being developed. Additional experiments are exploring the retargeting of insulated vectors to transcriptionally inactive chromatin through use of chimeric integrases.21Artus A Duros C Botbol Y Scholtz S Schmidt M von Kalle C et al.Directed integration of insulated lentiviral vectors to the heterochromatin towards safer gene transfer to stem cell (abstract 389).Presented 20 May 2011 at the Annual Meeting of the Society of Gene and Cell Therapy, Seattle, WA. 2011Google Scholar Frederic Bushman gave an update on data his laboratory has accumulated from studies of integration sites using high-throughput 454 sequencing of sequences flanking HIV-1 integration sites. Integrase determines viral integration–site preference, as demonstrated by studies in which substitution of the MLV integrase into HIV-1 vectors resulted in integration profile