Gene Therapy (2004) 11, S3–S9 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt REVIEW Retroviral vectors: new applications for an old tool

J Barquinero, H Eixarch and M Pe´rez-Melgosa Unitat de Diagno`stic i Tera`pia Molecular, Centre de Transfusio´ i Banc de Teixits, Psg. Vall d’Hebron 119–129, 08035 Barcelona, Spain

Retroviral vectors (RVs) have been used for stable gene development of T-cell acute leukaemia in two of 10 children transfer into mammalian cells for more than 20 years. The participating in one of these clinical trials, demonstrate the most popular RVs are those derived from the Moloney great potential of RVs, but also some potential risks which murine leukaemia virus (MoMLV). One of their main may be intrinsically associated with their use. Basic aspects limitations is their inability to transduce noncycling cells. of RVs and vector production were reviewed in detail in a However, they have a relatively simple genome and previous supplement of this journal. This article will first structure, are easy to use, and are relatively safe for in vivo summarize some general aspects of retroviruses and RVs. applications. For the last two decades, the artificial evolution Thereafter, recent developments in gene therapy using RVs, of RVs has paralleled evolution in their applications, which novel applications such as stable RNA interference and now include those as diverse as the generation of transgenic some other recent issues related to retroviral integration, animals, the stable delivery of small interfering RNA (siRNA) including clonality studies after haematopoietic stem cell and gene therapy clinical trials. Recent reports of two transplantation, retroviral tagging and insertional oncogen- successful gene therapy clinical trials in patients with severe esis will be discussed. immunodeficiency disease in France and Italy, and the Gene Therapy (2004) 11, S3–S9. doi:10.1038/sj.gt.3302363

Keywords: retroviral vectors; retroviruses; applications; insertional mutagenesis; insertional oncogenesis

Retrovirus and retroviral vectors: an overview that the first two successful gene therapy clinical trials, on X-linked severe immunodeficiency disease (SCID-X1) For the last two decades, retroviral vectors (RVs) have and ADA deficiency, were based on HSCs and used been major players in the fields of gene transfer and gene RVs.4,5 Subsequent reports of development of T-cell therapy.1 In the early 1980s, they were the first genetic leukaemia in two of 10 children treated with this vectors to permit an efficient and stable gene transfer experimental therapy in France6 were totally unexpected into mammalian cells.2 In 1990, RVs were the first vectors and will be discussed in more detail later in this article. used in a gene therapy clinical trial (for adenosine RVs are derived from retroviruses, lipid-enveloped deaminase (ADA) deficiency).3 In 2000, after a decade of particles containing two identical copies of a linear hopes and relative frustration, RVs were used in the first single-stranded RNA genome of around 7–11 kb. They successful protocol that actually cured a genetic disease, usually require binding to a specific membrane-bound demonstrating proof of concept for gene therapy.4 In all receptor for viral entry. Cells not expressing the appro- these years, vectors based on the Moloney murine priate receptor are resistant to infection by a specific leukaemia virus (MoMLV) have been pivotal in thou- retrovirus. In cytoplasm, viral reverse transcriptase retro- sands of experiments, and continue to constitute the best transcribes the viral genome into double-stranded DNA tool available for stable gene transfer into a number of (dsDNA), which binds to cellular proteins to form a cell types and applications. nucleoprotein preintegration complex (PIC), which con- Keys to their enormous success include the relative tains karyophilic elements that facilitate its migration to simplicity of their genomes, ease of use and their ability the nucleus. Nuclear membrane is a physical barrier for to integrate into the cell genome, permitting long-term these PICs. The size and ability of these PICs to migrate transgene expression in the transduced cells or their to the nucleus determine their capacity to reach the progeny. These characteristics render them ideal vectors cellular genome and transduce quiescent cells. In fact, for for a stable correction of genetic defects. In this regard, most retroviruses, such as MoMLV, PIC cannot cross stem cells in general, and haematopoietic stem cells the nuclear membrane, which renders these viruses and (HSCs) in particular, constitute optimal targets for RV- vectors incapable of infecting cells unless they undergo mediated gene transfer, since transgenes can be ex- a mitotic cycle which actually disrupts the nuclear pressed long term in vivo and may give rise, throughout a membrane. HIV-1 does not have this limitation, and is continuous amplification process, to a large progeny of capable of transducing quiescent cells, although trans- gene-modified mature cells. In fact, it was not a surprise duction efficiency by lentiviral vectors (LVs) is much higher in dividing cells than in nondividing cells. Correspondence: J Barquinero, Unitat de Diagno`stic i Tera`pia Molecular, Retroviruses infect birds and mammals, where they tend Centre de Transfusio´ i Banc de Teixits, Psg. Vall d’Hebron 119–129, 08035 to establish chronic infections, which are usually latent Barcelona, Spain and well tolerated for long periods of time and New applications of retroviral vectors J Barquinero et al S4 eventually result in the development of immunodefi- vectors contain a deletion of 299 bp in the 30 LTR, which ciency or malignancy. The retroviridae family consists blunts the functionality of the enhancer and the of seven genera: alpharetrovirus (whose type species is promoter. During reverse transcription, the deletion is the Avian leucosis virus), betaretrovirus (Mouse mam- transferred to the 50 LTR, resulting in the transcriptional mary tumour virus), gammaretrovirus (Murine leukae- inactivation of the provirus in the infected cell. SIN mia virus), deltaretrovirus (Bovine leukaemia virus), vectors require an internal promoter to drive transgene epsilonretrovirus (Walleye dermal sarcoma virus), lenti- expression, which permits the use of inducible or tissue- virus (Human immunodeficiency virus 1) and spuma- specific promoters to regulate gene expression. Many virus (Human spumavirus).7 The first five genera were RVs and most LVs currently used are SIN. Additionally, formerly classified as oncoretrovirus. Although, strictly the absence of enhancer and promoter sequences in both speaking, vectors based on lentivirus and spumavirus LTRs of the integrated provirus minimizes the risk of or foamy virus (FVs) are also RVs, this term is often used insertional oncogenesis, thus providing a safer alterna- to refer to vectors based on the MoMLV or the former tive for human gene therapy. oncoretrovirus. Although the inspiration to use viral vectors for human gene therapy probably originated in the early 1970s,8,9 the knowledge and tools to engineer viral genomes did not sufficiently develop until some Novel applications of RVs years later. The idea underlying the generation of RVs was conceived independently by three researchers in the Gene therapy for immunodeficiencies: early 1980s, Edward M Scolnick at the NIH, Howard the first successes, at last Temin at the University of Wisconsin at Madison and The ultimate challenge for RVs is gene therapy. Since the Robert Weinberg at the Massachusetts Institute of beginning of the first gene therapy clinical trials in the Technology, who observed that retroviruses could pick early 1990s, MoMLV-based vectors have been the up normal cellular genes from animal cells during preferred tool for the permanent correction of genetic infection. defects. A total of 254 gene therapy clinical trials, which The large-scale production of RVs for clinical applica- account for 28% of the total clinical trials approved tions still faces several challenges such as high titre worldwide, have relied on this type of vector (http:// vector production and the potential generation of www.wiley.co.uk/genmed/clinical). replication-competent particles. Improving vector de- Primary immunodeficiencies are ideal candidates for signs, finding strategies for cell targeting, understanding stem cell gene therapy for a number of reasons: the stem cell biology, achieving regulated and tissue-specific potential selective advantage for the gene-corrected cells expression, modulating factors determining transgene and the theoretical lack of immune responses to vector expression and silencing phenomena or controlling system components or transgene products. These are immune responses to the transgene products constitute good reasons to explain why they were the first diseases important issues that must be resolved if RVs are to be in which gene therapy succeeded. In some respect, gene widely applied to treat human disease. In recent years, therapy clinical trials for SCID can be considered the significant improvements in vectorology resulted in the optimum test bench for gene therapy for other diseases. generation of novel RVs with enhanced abilities and a The first milestone in clinical gene therapy was greater potential for gene therapy applications. One of reported by Cavazzana-Calvo et al (2000)4 in children these advances consisted of engineering the regulatory with SCID-X1. The disease is characterized by a defect in regions of RV to enhance transgene expression or reduce the common gamma chain (gc) gene, which encodes a transcriptional silencing in specific target cells. This led subunit of the receptors for interleukins 2, 4, 7, 9, 15 and to the generation of novel families of vectors such as the 21. SCID-X1 accounts for approximately half of all SCID FMEV, which contains hybrid regulatory sequences,10 or cases. Children with the disease have an almost complete MND, which contains an engineered LTR derived from absence of functional B, T and NK cells, which leads to the myeloproliferative sarcoma virus.11 These vectors severe and recurrent infections that are generally fatal provide higher levels of transgene expression in haema- during the early years of life. The gene therapy clinical topoietic progenitors than conventional RVs. trial conducted by Fischer and co-workers involved The use of heterologous envelopes (env) from other retrovirally mediated transfer of the gc gene into the viruses in RV production (pseudotyping) was a further SCID-X1 patients’ own marrow cells, which were major advance. RVs pseudotyped with the env of the subsequently reinfused back into the patients in the gibbon ape leukaemia virus (GALV) or the feline absence of any myelosuppressive treatment. A few endogenous retrovirus (RD114) transduced HSCs more months after infusion of the gene-corrected marrow efficiently than amphotropic RVs.12,13 The G protein from cells, T-cell counts were restored to almost normal levels the vesicular stomatitis virus (VSV-G) can also be used to and immune function was significantly improved in nine pseudotype RVs. Since VSV-G is a fusogenic protein that of 10 children treated. These results were confirmed interacts with membrane phospholipids to facilitate viral independently by researchers in the UK using a similar entry, RVs pseudotyped with VSV-G do not require a protocol in six additional children. The key to the success cellular receptor for cell entry, which permits a very of this landmark achievement is probably the fact that broad spectrum of infectivity.14 As an additional advan- gene-modified cells, able to respond to appropriate tage, RVs pseudotyped with VSV-G or RD114 are growth factors, have the selective advantage of growing physically more stable and can be concentrated by and repopulating an empty compartment (T cells), ultracentrifugation. whereas most noncorrected cells undergo apoptosis. A third improvement in RV technology was the The final result is an almost complete repopulation by development of self-inactivating (SIN) vectors.15 SIN functional immune cells and normalization of most

Gene Therapy New applications of retroviral vectors J Barquinero et al S5 immune parameters. This is generally accepted as the also suggests a selective advantage for ADA-expressing first genetic disease actually cured using gene therapy. myeloid cells.18 The second major success in gene therapy was These first successful achievements in human gene reported in 2002 in children with deficiency of the therapy bring new hope to the field. The fact that two enzyme ADA.5 ADA deficiency represents approxi- forms of SCID were the first genetic diseases to be cured mately 10–20% of all SCIDs. The enzyme catalyses the by gene therapy was not a surprise. The strong selective deamination of deoxyadenosine to adenosine, and its advantage for gene-corrected cells to proliferate or deficiency results in an accumulation of metabolites escape apoptosis is essential to explain this success. highly toxic for T cells, and it is clinically manifested as Unfortunately, this is not going to be the case for the a SCID. As occurs in SCID-X1, bone marrow transplant majority of candidate diseases that can potentially be from a matched sibling is the best treatment, if available. corrected at stem cell level. Several other forms of SCID, If not, patients can be treated with pegylated bovine now in the pipeline for gene therapy clinical trials at ADA (PEG-ADA), which partially corrects the immune different stages of preclinical development, include deficiency in the majority of patients. As mentioned Wiskott–Aldrich syndrome,19 JAK3 deficiency,20 RAG-1 above, the first human gene therapy clinical trial was and RAG-2 deficiencies21 and Artemis deficiency. carried out in children with ADA deficiency in 1990. The therapy consisted of using an MoMLV-based vector to Stable siRNA delivery insert a functional version of the ADA gene into the RNA interference (RNAi) is the phenomenon of se- patient’s own T cells, which were subsequently reinfused quence-specific, post-transcriptional gene silencing, eli- back into the patients. The two children treated, who cited by small double-stranded RNAs (dsRNA) that are were maintained on PEG-ADA therapy, still have homologous in sequence to the silenced gene.22 Many marked T cells in their peripheral blood, albeit at low experts consider RNAi the most useful tool for reverse levels. For many years, this clinical trial was considered genetics in the postgenomic era. Administration of unsuccessful, probably because ADA replacement ther- synthetic duplexes of 21–23 nucleotide RNAs was found apy was blunting the selective advantage for the gene- to mediate gene-specific RNAi in mammalian cells in modified cells. However, detailed analysis of the two vitro, without eliciting antiviral defence mechanisms.22 patients who received the therapy, almost 14 years ago, The use of these small interfering RNAs (siRNAs) revealed that in one patient, ADA activity was approxi- constitutes a powerful tool to dissect gene function. mately 25% of the normal level, with more than 15% of Administration of these molecules can inhibit target gene his peripheral blood mononuclear cells (PBMCs) still expression both in vitro and in vivo. However, the limited carrying the therapeutic gene. In the second patient, the lifespan of siRNAs in vivo and the short life of this effect results were far less satisfactory, with under 5% of the prompted several groups to develop vector-based sys- normal ADA activity and less than 0.1% of the gene tems for specific siRNA delivery into mammalian cells. marking level in PBMCs.16 A second gene therapy Efficient delivery and stable expression in target cells strategy, using autologous CD34+ cells from umbilical and their progeny necessarily requires the use of cord blood, instead of mature T cells, of prenatally integrative vectors. Both types of vectors, RVs and LVs, diagnosed children also resulted in suboptimal levels of have been reported to mediate an efficient and stable gene marking (1–10% of the circulating T cells) and little siRNA expression.23,24 Typically, the vectors use a pol III clinical benefit.17 These patients were also maintained on promoter, such as the U6, H1 or tRNA promoters. Vector replacement therapy with PEG-ADA, which might have systems for siRNA expression can be classified in two accounted for the limited success. A clear indication that main groups depending on whether the expressed RNAs gene-corrected cells have a selective advantage in this are hairpin-type or tandem-type. The former transcribe disease, and that PEG-ADA treatment actually blunted hairpin RNAs, which are subsequently processed into this selection, is that gradual discontinuation of the siRNA duplexes by endogenous RNase III. The tandem- therapy was associated with an increase in the percen- type siRNA-expression vectors include both sense and tage of marked peripheral blood T cells from a stable antisense sequences, which are transcribed and subse- level of 1–3% to virtually 100%, with a parallel increase quently annealed to generate the siRNA duplexes. in absolute T-cell counts and their functionality. The Using an MoMLV-based vector encoding an siRNA relative disappointment of these early studies resulted, specific for the p53 gene and a truncated form of human however, in the generation of highly useful information CD4 as a reporter gene, p53 expression was dramatically for subsequent gene therapy clinical trials. In 2002, reduced in cultured cells.25 In another report by researchers in Milan reported the results of their clinical Rubinson et al,26 LVs encoding an siRNA significantly trial on ADA deficiency. In this trial, autologous CD34+ reduced CD8 expression in vitro. In addition, those cells were transduced with an RV similar to that used in investigators also showed that siRNA was able to inhibit previous studies. However, a sublethal dose of busul- transgene expression in vivo in an EGFP transgenic phan was given to the patients as a partially myelo- mouse model. ablative treatment prior to the infusion of the gene-corrected cells. Moreover, no PEG-ADA was Clonality studies in the haematopoietic system administered after cell infusion. After several months, a Each single retroviral integration or insertion site (RIS) in significant improvement in immune parameters was the genome of HSC constitutes a unique marker that is observed in virtually all patients treated thus far, except also transmitted to their progeny, thus turning RISs into in one who still has subtherapeutic reconstitution levels. distinctive clonal tags within the haematopoietic system. Strikingly, a significant marking was also observed in the In the fields of gene therapy and stem cell biology, myeloid compartment, with up to 20% of marking in analysis of these RISs has two major applications: stem granulocytes, monocytes and megakaryocytes, which cell tagging for clonality studies and investigation of the

Gene Therapy New applications of retroviral vectors J Barquinero et al S6 vector insertion site preferences in the genomes of target gene-rich regions.35 In addition, they may also prefer cells. A number of molecular techniques have been some chromosomes to others. Laufs and co-workers used applied or specifically developed to analyse these RISs. retrovirally transduced human PBSCs transplanted into Southern blot analysis of restriction fragments containing immunodeficient mice to analyse RISs in the haemato- vector sequences permitted clonal characterization of poietic progeny generated in recipient mice 8 weeks after murine and human haematopoiesis after transplantation transplantation. The authors found that, although most of retrovirally transduced marrow cells.27–29 However, of the chromosomes were targeted, RV insertions the introduction of PCR-based techniques affords a more occurred relatively more often in chromosomes 17 and precise characterization and mapping of RISs, since the 19 and in specific regions of chromosomes 6, 13 and 16.31 amplified products containing the sequences flanking the Regarding lentiviral insertion preferences, a recent report proviral vector can be sequenced and mapped by in silico by Schroder et al characterized 524 RISs, after a single alignment search in mouse or human genome databases. round of HIV infection in a human lymphoid cell line. These techniques include inverse PCR30 and, more Almost 70% of the RISs analysed were found within recently, ligation-mediated PCR (LM-PCR),31 two-step genes.36 In a more recent report, 903 MoMLV and 379 PCR32 and linear amplification-mediated PCR (LAM- HIV-1 RISs were analysed in human cells. The study PCR),33 which is currently the most popular among stem showed that both RVs and LVs prefer to integrate close to cell researchers. genes; however, the authors found significant differences RIS analyses can be carried out in individual haema- between them. MoMLV-based vectors were more prone topoietic colonies that are, by definition, clonal. How- to insert close to the regulatory regions and the start of ever, the newer and more sensitive approaches such as transcriptional units (either upstream or downstream), LAM-PCR allow analysis of whole blood samples or whereas integrations of HIV-1-based vectors did not enriched phenotypically defined cell subpopulations show this preference and occurred anywhere in the such as neutrophils, T cells or NK cells. Since the transcriptional unit but not upstream of the start site.35 information they provide is at clonal level, these Another integrative virus, the adeno-associated virus techniques are unravelling questions that could not be (AAV) serotype 2, was recently reported to also prefer properly answered in the past. LAM-PCR was used to active genes for integration.37 analyse the clonal makeup of haematopoietic progeny Although viral insertion sites have been used for many in non-human primates transplanted with retrovirally years to identify potential oncogenes and signalling transduced HSCs. More than 80 different transduced pathways involved in cancer, recent advances, such as clones were detected in peripheral blood cells of these high throughput PCR-based insertion site cloning, the animals. Results of kinetic analyses of peripheral blood availability of genetically modified animals and comple- samples indicated that individual clones were continu- tion of the mouse genome project, have enormously ously contributing to generating specific haematopoietic widened the potential of this approach.38,39 A number lineages for several years.33 In another setting, this of investigators reported the identification of hundreds technique was used to analyse the clonal pattern of the of common integration sites (CISs, defined as insertion transduced haematopoietic progeny of ADA-deficient sites found in more than one tumour) associated with SCID children undergoing stem cell gene therapy. In newly identified cancer genes in MoMLV-induced these patients, T-cell reconstitution by gene-modified murine haematopoietic malignancies.40–42 In general, HSCs was found to be more oligoclonal than that of the the vast majority of insertions were found outside the previous report. In one patient, a single vector integrant coding regions. Less than 10% of the RISs actually was clearly predominant in T cells. At 8 years post- disrupted genes, which can thereafter be considered transplantation, isolated T cells with this prevailing putative tumour suppressor genes. Interestingly, ap- integrant showed multiple patterns of T-cell receptor proximately 17–18% of ISs targeted transcription factors. (TCR) gene rearrangement, thereby indicating that a All this information is now freely available at the single progenitor cell can generate the majority of the Retroviral Tagged Cancer Gene Database (RTCGD, gene-marked cells over very long periods of time.34 In online access at http://RTCGD.ncifcrf.gov).43 The data- another study, analysis of the clonal dynamics of retro- base, which contains more than 3100 RISs and more than virally transduced human HSCs grafted into NOD/SCID 230 CISs from near to 1000 retrovirally induced haema- mice, including transplantation into secondary recipi- topoietic tumours, manages several high-throughput ents, revealed that the stem cell compartment is highly insertional mutagenesis screening projects and contains heterogeneous and contains different types of repopulat- all the information available on RISs reported in murine ing cells, some contributing to short-term repopulation leukaemias and lymphomas. and others to long-term repopulation.29 By definition, all insertions of genetic material into a cellular genome are mutagenic. However, the risk of insertional oncogenesis after standard gene transfer Inserting tags in cancer pathways: retroviral tagging procedures using integrative vectors in preclinical and insertional oncogenesis animal studies, although sporadic cases have been Until recently, very little was known regarding retroviral reported,44 was believed to be very low.45 For this reason, integration selectivity. Recent reports on insertional the finding that two of the 10 children treated in the mutagenesis have renewed interest in investigating French clinical trial for SCID-X1 developed T-cell vector insertions. RVs were believed to integrate ran- leukaemia due to single vector insertions, in both cases domly in the genomes of host cells. Today, it is widely near the regulatory region of the LMO2 gene, was accepted that integration of retroviruses and RVs is extremely shocking and unexpected. In recent months, a skewed. RIS mapping studies have clearly shown that host of new information has accumulated that may retroviruses have a striking integration preference for finally translate into a clear idea of what caused these

Gene Therapy New applications of retroviral vectors J Barquinero et al S7 two severe adverse effects (SAEs). This is crucial for the the gene-modified cells. Both were the youngest patients future of gene therapy so that the risks associated with treated in the French trial. A younger age might be a specific therapy or procedure can be minimized. associated with a different gene expression profile in Common sense tells us that individual observations HSCs, a different proportion of HSCs subsets or an apparently contradicting a large body of knowledge are increased predisposition to oncogenic transformation. probably exceptions to a general rule. The occurrence of Another potential factor that may have contributed T-cell leukaemia in two children undergoing the same to development of the leukaemia is the high number of gene therapy procedure, and the discovery that the same actively proliferating lymphoid progenitors that were oncogene was probably involved in both SAEs, argued collected from these patients and subsequently trans- against the notion that risks of insertional oncogenesis duced and transplanted. This situation, which is prob- for these procedures were very low. However, similar ably specific for this particular disease, may have SAEs had not previously been reported, either around increased the number of progenitor cells potentially 200 patients treated so far with analogous gene therapy predisposed to dangerous vector insertions. Addition- protocols for other diseases using integrative vectors, ally, the tremendous selective pressure of the gene- including ADA deficiency, or, albeit anecdotally, in the corrected progenitor cells to repopulate a virtually empty thousands of experimental animals undergoing pre- compartment in vivo (1000–10 000-fold expansion) may clinical gene transfer studies conducted to date.45 This also have played a role.46 raised the crucial issue of whether gene therapy using A second major question was whether disease- and/or integrative vectors was much more dangerous than protocol-specific factors might have played a role in the previously thought, or whether these two cases were development of the leukaemias. In this regard, one more related to context-dependent factors and this could possibility is the unregulated expression of the gc not be extrapolated to other settings. transgene itself, which is driven by the retroviral LTR. The question of why a retroviral insertion near the gc is a subunit of receptors such as IL2R which, upon same proto-oncogene was related to the two cases of activation, promotes T-cell proliferation and inhibits leukaemia was especially intriguing. There are at least apoptosis. Whether expression of the other components two hypotheses, mutually nonexclusive, to respond to of these receptors can limit or modulate the expression of this question. One postulates that RVs insert preferen- the functional receptor is not yet known. However, it is tially into specific hotspots, and the second supports the noteworthy that Hacein-Bey-Abina et al6 were not able to idea that there are no significant preferences for integra- detect any abnormalities of the gc-signalling pathway in tion near specific genes, but a strong selection of the patients’ leukaemic cells. Involvement of gc over- integrants carrying critical or synergistic oncogenic expression and signalling in the pathogenicity of these insertions, as previously reported in mice.39 The avail- leukaemias would be in agreement with the hypothesis able data clearly support the second hypothesis. of the multiple hit for cancer development. Also of LMO2, the gene targeted in the two cases of interest was the finding that leukaemic cells of the SCID- leukaemia, encodes a transcription factor which is X1 patients with insertional activation of LMO2 were considered as a central regulator of normal haematopoi- CD3+ with fully rearranged TCR,6 whereas T-cell clones esis. Abnormal expression of LMO2 is a frequent finding observed in children with ALL and LMO2 translocations in childhood T-cell acute lymphocytic leukaemia, mostly are usually CD3À, indicating that transformation occurs related to chromosomal translocations. Transgenic mice before TCR rearrangement. This suggests that a different overexpressing LMO2 are more prone to developing mechanism of transformation probably operates in both T-cell leukaemias. In humans, LMO2 is highly expressed types of LMO2-associated leukaemia. in bone marrow CD34+ progenitor cells, moderately Insertional oncogenesis was also reported in experi- expressed in immature thymic cells (CD34+ CD1aÀ) and mental animals receiving bone marrow cells transduced low or absent in more mature thymic cells (CD34+ with a recombinant retrovirus encoding truncated nerve CD1a+).6 growth factor receptor (NGFR). A total of 10 mice Among the potential factors that might have con- receiving a marrow graft of the same origin developed tributed to these SAEs, one must consider the high leukaemia. Analysis of the leukaemic cells showed that number of marrow cells grafted in the two children (both the RV was inserted within the Evi 1 gene, which was patients received the two largest grafts). It has been deregulated, and that the truncated and theoretically estimated that the odds of an insertion in a given gene defective NGFR was in fact able to transduce a growth are B1 Â10À5. This is considering insertions less than signal upon stimulation by its ligand.44 Retroviral 10 kbp away from the transcriptional units. However, insertional activation of the Evi 1 gene is one of the most their potential for transactivation is probably higher, common events associated with transformation in since the retroviral enhancer has been reported to murine myeloid leukaemia. The role of the truncated transactivate genes over much longer distances (up to NGFR in the leukaemic transformation remains con- 100 kbp). If we assume that each patient received at least troversial, as recently observed by Bonini et al. These 106 transduced progenitor cells, the grafts must have authors were not able to detect any transformation included, on average, 1–10 progenitor cell clones carry- events in a series of experiments using this transgene.47 ing insertions near the LMO2 locus.6 This has significant Early this year, Dave´ et al, at the National Cancer repercussions in determining the optimal graft size for Institute (USA), unveiled a key information that could these patients, since larger grafts are likely to be finally explain the development of leukaemia. By associated with higher chances of containing cells with searching their above-mentioned Mouse Retroviral dangerous insertions than smaller ones. Another poten- Tagged Cancer Gene Database, they found two murine tial contributing factor could have been the young age of leukaemias associated with insertions at the LMO2 gene the children (1 and 3 months) at the time they received locus, and two with insertions at the IL2RG gene locus.

Gene Therapy New applications of retroviral vectors J Barquinero et al S8 One of them actually contained insertions at both loci.48 and-paste transposition in mammalian cells in vitro and The chances of these two unlikely events occurring in the in somatic tissues or the germ line of the mouse in vivo, same cell are extremely remote. This new finding further and have already proved to be able to mediate gene supports the notion that the leukaemic transformations transfer at clinically significant levels.54 were context dependent in these two children. This could As for RVs, they may still have a lot to say in the be very good news for the field and may finally help future. At present, no other vectors can substitute them to restore the image of RVs and confidence in gene for most gene therapy applications. However, most therapy.49 researchers agree they must be improved. There is much The two cases of leukaemia reported by the French room for improvement, including better vector designs, researchers triggered rapid unintentional responses by systems for conditional expression, use of selectable the media and society, and scepticism by the scientific genes, use of insulators to eliminate surrounding community. Several regulatory agencies in many coun- promoter effects of the retroviral enhancer or, alterna- tries banned gene therapy protocols based on integrative tively, use of SIN vectors in conjunction with safer tissue- vectors, and some important companies even reoriented specific promoters. Regarding clinical trials, each vector their lines of research away from RV. Altogether, these system will have to be carefully validated on a case-by- unfounded reactions became a pessimistic wave of case basis. It will take some time for gene therapy to be opinion at several levels, and led to the paradox that better understood and for tools to be improved; however, children with SCID-X1 were not allowed to receive a there is no doubt that, currently, it constitutes the best therapy that could save their lives. It is worth mention- weapon and the only hope to cure genetic diseases. ing that unrelated bone marrow transplantation, a therapy widely accepted by society and regulatory agencies and which constitutes the only alternative therapy for these children, has a mortality rate of around Acknowledgements 30%, a much higher risk than that of gene therapy- We thank Ana Limo´n, Robert Tjin and Christine O’Hara induced leukaemia in this disease (two of 16 cases for critical review of the manuscript. The group is treated worldwide). In addition, survivors of this supported by grants from the Ministerio de Ciencia y procedure often have to face other medical complications Technologia FIS, the Spanish Collaborative Network on such as graft-versus-host disease or an increased risk of Transplantation and the Vth Framework Programme of lymphoma. This posed a great ethical dilemma, since the European Commission (http://www.inherinet.org). gene therapy for SCID was still the best therapeutic option for these children. The facts are that current gene therapy protocols for SCID-X1 in Europe have saved 15 of 16 patients treated (10 in France and six in the UK), References and that both children developing leukaemia subse- quently underwent an unrelated marrow transplant and 1 Pages JC, Bru T. Toolbox for retrovectorologists. J Gene Med 2004; 6: S67–S82. are currently in remission. At present, many countries 2 Cone RD, Mulligan RC. 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