Lentiviral Vector Gene Therapy: Effective and Safe? (2010)

Over the past decade, more than 30 patients with different immunodeficiency disorders have been treated successfully using murine leukemia virus (MLV)-based γ-retroviral vectors to transfer therapeutic genes to autologous hematopoietic cells.1Aiuti A Roncarolo MG Ten years of gene therapy for primary immune deficiencies.Hematology Am Soc Hematol Educ Program. 2009; 288: 682-689Google Scholar,2Cavazzana-Calvo M Hacein-Bey S de Saint Basile G Gross F Yvon E Nusbaum P et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (2242) Google Scholar,3Aiuti A Slavin S Aker M Ficara F Deola S Mortellaro A et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (983) Google Scholar However, this approach has been complicated by adverse events caused by γ-retroviral vector integration into the target cell genome. Five cases of lymphoid leukemia have been reported from two different X-linked severe combined immunodeficiency (SCID-X1) trials,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 (3010) Google Scholar,5Howe SJ Mansour MR Schwarzwaelder K Bartholomae C Hubank M Kempski H 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 (952) Google Scholar while a recent report now documents two cases of myelodysplasia occurring in a clinical trial for X-linked chronic granulomatous disease.6Stein 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 (634) Google Scholar A common finding in these patients was transcriptional activation of nearby proto-oncogenes by the powerful enhancer elements contained within the γ-retroviral long terminal repeats (LTRs) of the vector—a feature that has been difficult, in the case of SCID-X1, to recapitulate in animal models (see the Commentary by Sorrentino in this issue7Sorrentino B Assessing the risk of lymphomagenesis in gene therapy for SCID-X1: a mouse model shows transformation without vector-induced oncogene activation.Mol Ther. 2010; 18: 868-870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). These events fostered the development of several new in vitro and in vivo models to study vector genotoxicity. One study reported the apparent enhanced safety of lentiviral vectors based on HIV.8Montini E Cesana D Schmidt M Sanvito F Ponzoni M Bartholomae C et al.Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.Nat Biotechnol. 2004; 24: 687-696Crossref Scopus (586) Google Scholar Two major factors seem to be important for the initial observation of decreased genotoxicity of lentiviral vectors, relative to a standard γ-retroviral vector. The first is that lentiviral vectors were effectively developed early on without viral LTR enhancer elements and that these so-called self-inactivating (SIN) vectors could be made without loss of titer. In contrast, early versions of the comparable SIN γ-retroviral vectors exhibited low titers and were thus not extensively pursued for clinical application. Thus, γ-retroviral vectors with intact LTRs were utilized in the early trials. Second, the overall insertion site selection of lentiviral vectors is different from that of γ-retroviral vectors, probably due in part to differences between the integrases of the two type of viruses and the presence of the viral LTRs in the γ-retroviral vectors.9Montini 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 (463) Google Scholar,10Felice B Cattoglio C Cittaro D Testa A Miccio A Ferrari G et al.Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.PLoS One. 2009; 4: e4571Crossref PubMed Scopus (85) Google Scholar Although lentiviral vectors target active transcription units, they integrate somewhat randomly throughout the entire gene. In contrast, the integrase and LTRs of γ-retroviral vectors seem to promote preferential interaction with active host cell promoters and enhancers—regions that are enriched for transcription factor binding sites (TFBS).10Felice B Cattoglio C Cittaro D Testa A Miccio A Ferrari G et al.Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.PLoS One. 2009; 4: e4571Crossref PubMed Scopus (85) Google Scholar Because many cancer-related genes are often highly expressed in primitive hematopoietic cells and thus are rich in such regions, this might at least partly explain differences in the safety profiles of the two vector types. Therefore, the first report of a clinical trial utilizing an HIV-based, lentiviral vector has been eagerly awaited from the standpoints of both efficacy and safety. A few months ago, Cartier et al. reported the successful treatment of two children with X-linked adrenoleukodystrophy (X-ALD) using a lentiviral vector encoding the deficient peroxisomal adenosine triphosphate–binding cassette transporter protein ABCD1 (ref. 11Cartier 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 (1182) Google Scholar). Nearly 2 years after transplantation with transduced autologous CD34+ cells, 9–14% of peripheral blood cells of the patients express the ABCD1 protein and neurological function has stabilized. An extensive analysis of vector insertion sites thus far shows a highly polyclonal hematopoiesis by the transduced cells. Importantly, despite the use of a lentiviral vector that contained an internal viral LTR to drive transgene expression, no clustering of vector insertions in oncogenes or growth-related genes was observed. This contrasts with the γ-retroviral clustering in such sites that was noted even in SCID-X1 patients who did not develop malignancy or clonal dominance.12Deichmann A Hacein-Bey-Abina S Schmidt M Garrigue A Brugman MH Hu J et al.Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.J Clin Invest. 2007; 117: 2225-2232Crossref PubMed Scopus (219) Google Scholar,13Schwarzwaelder K Howe SJ Schmidt M Brugman MH Deichmann A Glimm H 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 (175) Google Scholar The clinical efficacy together with the pattern of vector insertions reported in this trial thus seemed quite encouraging for the use of lentiviral vectors. Unfortunately, a report of clonal dominance, despite clinical improvement, in a different clinical trial using a β-globin lentiviral vector has injected a note of caution into this early positive assessment. Although this study has not yet been formally published in a peer-reviewed format, we learned last summer—from a memo issued by the National Institutes of Health's Office of Biotechnology Activities, via public presentations by the investigators, and through a news article in Science—that a relative clonal dominance occurred in a patient treated with gene therapy for a form of β-thalassemia.14Kaiser J Gene therapy: beta-thalassemia treatment succeeds, with a caveat.Science. 2009; 326: 1468-1469Crossref PubMed Scopus (41) Google Scholar The patient was treated in June 2007 with autologous CD34+ cells transduced with a lentiviral vector encoding a variant β-globin gene transcribed from a promoter under the control of a strong erythroid enhancer. The dominant clone that emerged approximately a year after transplant contained an activating insertion within the HMGA2 proto-oncogene. Aberrant expression of HMGA2 was likely caused by the erythroid enhancer carried by the vector. Although this clone comprises approximately one half of the progeny of the transduced cells, its abundance has been stable for many months since being discovered, implying that the clone is benign. Importantly, the patient is transfusion independent and exhibits no evidence of hematologic abnormality. At this point, cause, effect, and clinical significance remain unclear and the field awaits more details in a peer-reviewed format about this important trial and its implications for lentiviral vector safety. It seems reasonable to conclude for now that we need results from additional patients treated with lentiviral vector gene transfer before we can draw solid conclusions regarding the relative safety of this vector system. In this regard, two new upcoming trials for SCID-X1 that have recently undergone review by the Recombinant DNA Advisory Committee should prove highly informative (http://oba.od.nih.gov/oba/RAC/meetings/dec2008/RAC_Minutes_12-08.pdf; http://oba.od.nih.gov/oba/RAC/meetings/mar2009/March%202009%20minutes.pdf). Both trials propose the use of vectors devoid of viral LTRs or strong enhancers, which clearly elevate genotoxic risk. Building on recent improvements in the design of SIN γ-retroviral vectors that can be produced with effective titers,15Schambach A Mueller D Galla M Verstegen MM Wagemaker G Loew R et al.Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.Gene Ther. 2006; 13: 1524-1533Crossref PubMed Scopus (119) Google Scholar one trial proposes the use of a SIN MLV-based γ-retroviral vector that utilizes the elongation factor (EF)-1α cellular promoter to direct transgene expression. Recent preclinical studies have shown that such a design results in a vector that compares favorably to a lentiviral vector in genotoxicity studies and gives rise to significantly reduced clustering of integration sites at TFBS-rich regions in the genome.10Felice B Cattoglio C Cittaro D Testa A Miccio A Ferrari G et al.Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.PLoS One. 2009; 4: e4571Crossref PubMed Scopus (85) Google Scholar,16Zychlinski D Schambach A Modlich U Maetzig T Meyer J Grassman E et al.Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol Ther. 2008; 16: 718-725Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar The second trial proposes to use a SIN lentiviral vector that incorporates an insulator element and also exploits the EF-1α promoter to drive gene expression. Comparisons of clinical outcomes and insertion-site profiles will be interesting and most assuredly instructive. Currently, the strategy of these trials to eliminate enhancer sequences from the vector would seem to be the best approach to minimize genotoxicity of vector integration. However, this is dependent upon the ability to achieve therapeutic transgene expression in the target cells using a promoter of relatively weak to moderate strength. For disorders in which high-level transgene expression is required for a therapeutic effect (e.g., β-thalassemia), inclusion of some type of enhancer element in the vector will be unavoidable. One potential solution to minimize the risk of such vectors is to target their integration to “safe” regions within the genomes. However, realization of this goal is years away. For now, the most practical solution is to include an effective insulator element (enhancer blocker) in the vector, which can prevent the interaction of the enhancer with nearby genes. Although some elements have been identified in various assays that reduce the risk of viral enhancer–mediated transcriptional activation of cellular genes, thus far none has shown the ability to do so absolutely. It is likely that an optimal insulator will need to be identified on a case-by-case basis for each specific enhancer-containing vector, taking into account the cellular context. This should be possible as methods advance to identify novel enhancer-blocking elements17Xi H Shulha HP Lin JM Vales TR Fu Y Bodine DM et al.Identification and characterization of cell type–specific and ubiquitous chromatin regulatory structures in the human genome.PLoS Genet. 2007; 3: e136Crossref PubMed Scopus (180) Google Scholar for testing in gene transfer models. Exciting days are ahead for hematopoietic cell–directed gene therapy. Assessing the Risk of T-cell Malignancies in Mouse Models of SCID-X1Brian SorrentinoMolecular TherapyMay, 2010In BriefIt has long been recognized that severe combined immunodeficiency disorders (SCIDs) are favorable settings for the application of stem cell gene therapy. This genetically heterogeneous group of disorders includes adenosine deaminase (ADA) deficiency and X-linked SCID (SCID-X1),1 the latter caused by mutations in the common gamma chain (γc) gene, which is required for a broad range of cell signaling events in lymphocytes and their precursors. The strong selective advantage for genetically corrected cells during development circumvents the requirement for high levels of transduction and engraftment, which remains a significant barrier for other blood diseases that lack an in vivo selection mechanism for transduced cells. Full-Text PDF Open Archive