Replication-competent Vectors and Empty Virus-like Particles: New Retroviral Vector Designs for Cancer Gene Therapy or Vaccines
Authors: Charlotte Dalba, Bertrand Bellier, Noriyuki Kasahara, David Klatzmann
Journal: Molecular Therapy (2007)

Replication-defective vectors based on murine oncoretroviruses were the first gene transfer vectors to be used in successful gene therapies. Despite this achievement, they have two major drawbacks: insufficient efficacy for in vivo gene transfer and insertional mutagenesis. Attempts to overcome these problems have led to two retroviral vector designs of principally opposite character: replication-competent vectors transducing largely intact genomes and genome-free vectors. Replication-competent retroviral vectors have achieved dramatically improved efficacy for in vivo cancer gene therapy and genome-free retroviral vectors expressing different kinds of antigens have proven excellent as immunogens. Current developments aim to improve the safety of the replication-competent vectors and to augment the production efficiency of the genome-free vectors by expression from heterologous viral or non-viral vectors. Together with the continuous advances of classical defective retroviral vectors for ex vivo gene therapy, these developments illustrate that, due to their tremendous design versatility, retroviral vectors remain important vectors for gene therapy applications. Replication-defective vectors based on murine oncoretroviruses were the first gene transfer vectors to be used in successful gene therapies. Despite this achievement, they have two major drawbacks: insufficient efficacy for in vivo gene transfer and insertional mutagenesis. Attempts to overcome these problems have led to two retroviral vector designs of principally opposite character: replication-competent vectors transducing largely intact genomes and genome-free vectors. Replication-competent retroviral vectors have achieved dramatically improved efficacy for in vivo cancer gene therapy and genome-free retroviral vectors expressing different kinds of antigens have proven excellent as immunogens. Current developments aim to improve the safety of the replication-competent vectors and to augment the production efficiency of the genome-free vectors by expression from heterologous viral or non-viral vectors. Together with the continuous advances of classical defective retroviral vectors for ex vivo gene therapy, these developments illustrate that, due to their tremendous design versatility, retroviral vectors remain important vectors for gene therapy applications. Retroviruses have been studied extensively for almost 100 years (Figure 1, see Supplementary Text for a brief retrospective of milestone events). This work has created a vast knowledge base of relevance to the development of the retroviral vectors, which initiated the development of gene therapy. Gene therapy involves the introduction of genetic material into a cell or tissue in order to (i) replace or repair a missing or defective gene to treat a genetic disorder, (ii) regulate the expression of genes for therapeutic purposes, or (iii) supplement a gene such as (a) a tumor suppressor or cytotoxin-activating gene to treat a malignancy or (b) an antigen-encoding or immunostimulatory gene to prevent or cure an infectious disease. Permanent modification of the host cell genome is the generally preferred outcome in the former case, whereas temporary regulation or complementation may suffice in the latter cases. In all cases, efficient and tissue-specific gene transfer as well as means to control the level and duration of gene expression are of paramount importance for efficacy and safety. Retroviruses have several distinct inherent advantages with respect to both their gene and protein make-up, which constitute keys to their success in gene therapy. These include (i) well studied genomes that are amenable to genetic engineering, (ii) sufficiently small genomes to allow their incorporation into and expression from various heterologous vectors, (iii) exclusive ability to transform their single-stranded RNA genome into double-stranded DNA that stably integrates into and permanently modifies the host cell genome, (iv) relatively low immunogenicity compared to some other viral vectors, and (v) the unique ability to incorporate envelope proteins from a wide range of heterologous viruses, providing a means for specific cell targeting or foreign antigen display. These features are of differing interest depending on the intended application and this review focuses on the main developments of retroviral vectors for application in "classical" gene therapy, cancer gene therapy, or genetic vaccines. Replication-defective retroviral vectors. The capacity of retroviruses to integrate into the host genome carries the risk of insertional mutagenesis and oncogene activation. Reducing this risk was a major goal of scientists designing the first retroviral vectors. To this end, replication-defective retroviral (RDR) vectors devoid of genes required for self-replication and thus incapable of cell-to-cell spread or general dissemination were developed.1Stuhlmann H Cone R Mulligan RC Jaenisch R Introduction of a selectable gene into different animal tissue by a retrovirus recombinant vector.Proc Natl Acad Sci USA. 1984; 81: 7151-7155Crossref PubMed Scopus (43) Google Scholar, 2Rapp UR Goldsborough MD Mark GE Bonner TI Groffen J Reynolds Jr, FH Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus.Proc Natl Acad Sci USA. 1983; 80: 4218-4222Crossref PubMed Scopus (317) Google Scholar, 3Cepko CL Roberts BE Mulligan RC Construction and applications of a highly transmissible murine retrovirus shuttle vector.Cell. 1984; 37: 1053-1062Abstract Full Text PDF PubMed Scopus (640) Google Scholar To decrease the potential emergence of replication-competent retroviral (RCR) vectors through inadvertent recombination events, overlapping sequences of the retrovirus genomes and those of the vector-producing cells were reduced to a minimum.4Miller AD Retrovirus packaging cells.Hum Gene ther. 1990; 1: 5-14Crossref PubMed Scopus (316) Google Scholar, 5Miller AD Rosman GJ Improved retroviral vectors for gene transfer and expression.Biotechniques. 1989; 7 (984–986, 989–990): 980-982PubMed Google Scholar, 6Yu SS Han E IIong Y Lee JT Kim s Kim S Construction of a retroviral vector production system with the minimum possibility of a homologous recombination.Gene Ther. 2003; 10: 706-711Crossref PubMed Scopus (27) Google Scholar Generally, RDR vector genomes consist of retrovirus genome cis sequences driving the expression of a therapeutic gene and include the packaging signal sequence necessary for their encapsidation (Figure 2). RDR vectors are produced in vitro by transient transfection or stable integration of RDR vector genomes in vector-producing cells containing stably integrated retrovirus genome trans sequences, encoding all of the structural elements necessary to assemble the vector particle (i.e., Gag, Pol, and Env), but lacking the packaging signal sequence necessary to encapsidate them (Figure 3). After infection of a target cell, the RDR vectors are able to integrate the genome sequence carrying the therapeutic gene, but are incapable of replication of secondary infection of adjacent cells. Indeed, the inability to replicate reduces the likelihood of unintended spread and mutagenic or oncogene-activating or tumor suppressor gene-inactivating insertion, and thus greatly improves the safety of these vectors. However, it also reduces their efficiency of gene delivery. Along these lines, these vectors have particular advantages and shortcomings depending on the application.Figure 3Schematic representation of retroviral vectors resulting from transfection of the RDR, RCR, s-RCR, or retroVLP vector genomes discussed in this review article. Transfection of RDR vector genomes into packaging cells containing stably integrated packaging genomes results in release of infectious but replication-incompetent viral particles that carry two copies of the RDR vector genome. Transfection of RCR vector genomes results in release of infectious replication-competent viral particles that carry two copies of the RCR vector genome. Transfection of s-RCR vector genomes results in release, from co-infected cells, of infectious viral particles that carry two copies of either the same or one of each of the trans-complimentary s-RCR vector genomes. Upon co-infection of a cell, the inherently replication-defective but trans-complimentary s-RCR vectors can replicate and give rise to new trans-complimentary s-RCR vectors. Transfection of retroVLP vector genomes results in release of genome-free replication-defective viral particles that are capable of cell entry through the cognate receptors of the envelope glycoprotein. The envelope glycoprotein may be a heterologous one and the envelope and/or core protein may express foreign epitopes of choice. Of note, vector genomes that cannot be reverse transcribed or integrated into the host genome exist 47Baum C Schambach A Bohne J Galla M Retrovirus vectors: toward the Plentivirus?.Mol Ther. 2006; 13: 1050-1063Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar but are not described in any detail in this review article.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The use of RDR vectors to deliver genes into hematopoietic stem cells ex vivo to cure two different forms of primary immunodeficiency, X-severe combined immunodeficiency, and adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID), long represented the only successes in human gene therapy clinical trials.7Gaspar HB Thrasher AJ Gene therapy for severe combined immunodeficiencies.Expert Opin Biol Ther. 2005; 5: 1175-1182Crossref PubMed Scopus (40) Google Scholar, 8Fischer A Iiacein-Bey-Abina S Lagresle C Garrigue A Cavazana-Calvo M Gene therapy of severe combined immunodeficiency disease: proof of principle of efficiency and safety issues. Gene therapy, primary immunodeficiencies, retrovirus, lentivirus, genome.Bull Acad Natl Med. 2005; 189 (Discussion 786–788): 779-785PubMed Google Scholar Several factors working in concert may explain why: (i) hematopoietic stem cells constitute optimal targets for retroviral vector-mediated gene transfer, as they can be manipulated with relative ease ex vivo, and transgenes can be expressed long-term in vivo by the stem cells' subsistence and generation of a large progeny of gene-modified mature cells, (ii) the relatively low immunogenicity of retroviral vectors and the immunodeficient status of the patients are likely to result in absent or minimal immune responses to vector components or transgene products, and (iii) the nature of the diseases causes a strong selective advantage for gene-corrected cells to proliferate or escape apoptosis. Unfortunately, selective advantage for growth may be linked not only to restored function induced by the transgene product, but also to inadvertent effects induced by the transgene insertion. This seems to be the case in the first X-severe combined immunodeficiency trial, where of the three treated children who developed leukemia-like clonal T-cell proliferation post-treatment, two died of complications.8Fischer A Iiacein-Bey-Abina S Lagresle C Garrigue A Cavazana-Calvo M Gene therapy of severe combined immunodeficiency disease: proof of principle of efficiency and safety issues. Gene therapy, primary immunodeficiencies, retrovirus, lentivirus, genome.Bull Acad Natl Med. 2005; 189 (Discussion 786–788): 779-785PubMed Google Scholar, 9Hacein-Bey-Abina S von Kalle C Schmidt M Le Deist F Wulffraat N McIntyre E et al.A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2003; 348: 255-256Crossref PubMed Scopus (1617) Google Scholar, 10Kohn DB Sadelain M Glorioso JC Occurrence of leukaemia following gene therapy of X-linked SCID.Nat Rev Cancer. 2003; 3: 477-488Crossref PubMed Scopus (309) Google Scholar In all cases, the provirus had integrated within or near the LMO2 oncogene.11Hacein-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. 2006; 302: 415-419Crossref Scopus (3010) Google Scholar It thus appears that activation of the LMO2 gene is particularly advantageous to the growth or survival of T cells and, in fact, its translocation is among the most prevalent of the translocations seen in T-cell leukemias.12Nam CH Rabbitts TH The role of LMO2 in development and in T cell leukemia after chromosomal translocation or retroviral insertion.Mol Ther. 2006; 13: 15-25Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Purely coincidental integration in or near this gene is unlikely. To a certain extent, there is evidence for genome region-specific integration of retroviruses13Cereseto A Giacca M Integration site selection by retroviruses.AIDS Rev. 2004; 6: 13-21PubMed Google Scholar, 14Wu X Burgess SM Integration target site selection for retroviruses and transposable elements.Cell Mol Life Sci. 2004; 61: 2588-2596Crossref PubMed Scopus (63) Google Scholar and it was recently shown that in these cases the vector integration occurred at a common fragile site, known to correlate with chromosomal breakpoints in tumors, attracting a non-random number of murine leukemia virus (MLV) integrations.15Bester AC Schwartz M Schmidt M Garrigue A Hacein-Bey-Abins S Cavazzana-Calvo M et al.Fragile sites are preferential targets for integrations of MLV vectors in gene therapy.Gene Ther. 2006; 13: 1057-1059Crossref PubMed Scopus (45) Google Scholar Finally, the inadvertent effects may have been induced by the transgene itself. Indeed, in a mouse model for X-severe combined immunodeficiency gene therapy, the corrective therapeutic gene itself could contribute to the genesis of T-cell lymphomas.16Woods NB Bottero V Schmidt M von Kalle C Verma IM Gene therapy: therapeutic gene causing lymphoma.Nature. 2006; 440: 1123Crossref PubMed Scopus (249) Google Scholar In the third successful gene therapy clinical trial that was recently reported, two adults suffering from X-linked chronic granulomatous disease attained notable clinical improvement after infusion of genetically modified hematopoietic stem cells.17Ott MG Schimdt M Schwarzwaelder K Stein S Siler U Koehi U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertionnal activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (984) Google Scholar Follow-up is ongoing to evaluate the long-term clinical outcome and malignant transformation, in line with lessons learned from the X-severe combined immunodeficiency trials. Altogether, RDR vectors deserve continued development. However, as we shall see below, certain diseases may benefit from retroviral vectors with quite different designs. Novel retroviral vectors—transducing all or nothing. In essence, gene therapy for genetic disorders and gene therapy for malignancies have two opposite goals; the former aims to restore a particular cell compartment and the latter aims to oust it. In gene therapy for genetic disorders, gene transfer to a subset of the target cells may suffice for restoration, but care must be taken that no inadvertent growth advantage linked to the gene transfer occurs, as the goal is life-long persistence of correctly modified target cells and their progeny. To the contrary, in gene therapy for malignancies, gene transfer to most if not all target cells is a requisite, but less attention may be given to inadvertent growth advantage, as the goal is to kill all transduced cells. Thus, vector modifications aimed at efficacy or safety may be given somewhat different priorities, depending on their intended application. As an example, in the largest clinical trial of RDR-mediated cancer gene therapy conducted to date, no more than 0.002% of the tumor cells were transduced in any examined patient,18Rainov NG Ren H Clinical trials with retrovirus mediated gene therapy—what have we learned?.J Neurooncol. 2003; 65: 227-236Crossref PubMed Scopus (89) Google Scholar which probably explains why no such protocol has been therapeutically adequate. Thus, for this particular application, retroviral vectors retaining their capacity for replication might be justified. As another example, the use of moderately replicating vectors (live attenuated virus) in vaccines, i.e., attenuated human immunodeficiency virus (HIV) to prevent HIV infection, has provided promising results,19Miller CJ Abel K Immune mechanisms associated with protection from vaginal SIV challenge in rhesus monkeys infected with virulence-attenuated SHIV 89.6.J Med Primatol. 2005; 34: 271-281Crossref PubMed Scopus (14) Google Scholar but such vectors are considered far too dangerous for use in healthy subjects. For this particular application, retroviral vectors empty of any genome represent a safer alternative. Likewise, many groups, including ours, have recently designed retroviral vectors that are either (i) fully replication competent for improved gene transfer, but which incorporate several alternative safety measures or (ii) completely devoid of any genome, but which incorporate foreign antigens of interest for specific immunogenicity. The remainder of this review will focus on the versatility of these two extremes of retroviral vector design. RCR vectors. The use of replication-competent vectors has seen growing interest particularly in the field of cancer gene therapy, where replication-defective vectors have mostly failed. This is because each tumor cell infected with a replication-competent vector becomes a vector-producing cell that gives rise to more vectors and thus more efficient transduction. Indeed, the use of replication-competent viruses as oncolytic agents has scientific precedent. In the early 1900s, it was discovered that chance infection with cytolytic viruses could efficiently inhibit or even reverse tumor growth.20Dock G Influence of complicating disease upon leukaemia.Am J Med Sci. 1904; 127: 563-592Crossref Google Scholar This discovery led to testing of the oncolytic power of various cytolytic viruses in the clinic. However, despite any initial tumor regression, the final result was often tumor recurrence resulting from this fact and the patients' anti-viral immune responses. Therefore, because of the contemporaneous advance of chemotherapeutic drugs, "oncolytic virotherapy" was largely abandoned. Since then, a better understanding of the molecular mechanisms involved in viral pathology and immunology, as well as improved means for precise manipulation of the gene or protein make-up of viruses, has allowed the engineering of replication-competent cytolytic viral vectors with more efficient oncolytic power.21Everts B van der Poel HG Replication-selective oncolytic viruses in the treatment of cancer.Cancer Gene Ther. 2005; 12: 141-161Crossref PubMed Scopus (130) Google Scholar, 22Davis JJ Fang B Oncolytic virotherapy for cancer treatment: challenges and solutions.J Gene Med. 2005; 7: 1380-1389Crossref PubMed Scopus (66) Google Scholar Among them, RCR vectors, which are not inherently cytolytic, have been developed as oncolytic agents.23Dalba C Klatzmann D Logg CR Kasahara N Beyond oncolytic virotherapy: replication-competent retrovirus vectors for selective and stable transduction of tumors.Curr Gene Ther. 2005; 5: 655-667Crossref PubMed Scopus (41) Google Scholar The first MLV-based RCR vectors were developed by insertion of either a transgene cassette into the U3 region of the 3′ long terminal repeat (LTR), allowing for LTR-driven transgene expression,24Dillon PJ Lenz J Rosen CA Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein.J Virol. 1991; 65: 4490-4493PubMed Google Scholar, 25Stuhlmann H Jaenisch R Mulligan RC Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.Mol Cell Biol. 1989; 9: 100-108Crossref PubMed Scopus (38) Google Scholar, 26Reik W Weiher H Jaenisch R Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.Proc Natl Acad Sci USA. 1985; 82: 1141-1145Crossref PubMed Scopus (66) Google Scholar or an additional splice acceptor site and transgene cassette downstream of the env gene, allowing for transgene expression following alternative splicing.27Solly SK Trajcevski S Frisen C Holzer GW Nelson E Clerc B et al.Replicative retroviral vectors for cancer gene therapy.Cancer Gene Ther. 2003; 10: 30-39Crossref PubMed Scopus (66) Google Scholar However, these configurations led to genetic instability. Generally, most recent RCR vector genomes consist of an intact retrovirus genome including an internal ribosome entry site-transgene cassette inserted immediately after the stop codon of the env gene27Solly SK Trajcevski S Frisen C Holzer GW Nelson E Clerc B et al.Replicative retroviral vectors for cancer gene therapy.Cancer Gene Ther. 2003; 10: 30-39Crossref PubMed Scopus (66) Google Scholar, 28Jespersen T Duch M Carrasco ML Warming S Pedersen FS Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors.Gene. 1999; 239: 227-235Crossref PubMed Scopus (27) Google Scholar, 29Logg CR Logg A Tai CK Cannon PM Kasahara N Genomic stability of murine leukemia viruses containing insertions at the Env-3′ untranslated region boundary.J Virol. 2001; 75: 6989-6998Crossref PubMed Scopus (61) Google Scholar (Figure 2), thus comprising all elements necessary for replication and RCR vector production following cell transfection (Figure 3). In in vitro studies, a small number of such RCR vectors achieved a tremendous amplification of transduced cells, as compared to RDR vectors27Solly SK Trajcevski S Frisen C Holzer GW Nelson E Clerc B et al.Replicative retroviral vectors for cancer gene therapy.Cancer Gene Ther. 2003; 10: 30-39Crossref PubMed Scopus (66) Google Scholar, 30Logg CR Tai CK Logg A Anderson WF Kashara N A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors.Hum Gene Ther. 2001; 12: 921-932Crossref PubMed Scopus (72) Google Scholar, 31Chang Z Pan J Logg C Kasahara N Roy-Burman P A replication-competent feline leukemia virus, subgroup A (FeLV-A), tagged with green fluorescent protein reporter exhibits in vitro biological properties similar to those of the parental FeLV-A.J Virol. 2001; 75: 8837-8841Crossref PubMed Scopus (10) Google Scholar and in one study they proved genetically stable when propagated over seven serial cell-free passages.29Logg CR Logg A Tai CK Cannon PM Kasahara N Genomic stability of murine leukemia viruses containing insertions at the Env-3′ untranslated region boundary.J Virol. 2001; 75: 6989-6998Crossref PubMed Scopus (61) Google Scholar However, murine tumor models have faster growth kinetics than many human tumors and, therefore, the efficacy of RCR vectors in murine models needs to be interpreted with caution. Also, although recent RCR designs show significant improvement over previous designs, the genetic instability of RCR vectors, i.e., the possible loss or incorporation of genetic sequences that may render the vectors inactive or give rise to chimeric vectors with unknown functions, must be kept in mind and deserves further study. Beyond the hallmark stable integration of retroviruses, RCR vectors have two features of particular interest for oncotherapeutics. First, they can only integrate and replicate in dividing cells, providing an inherent means for targeting malignant cells that divide rather actively, while sparing surrounding normal cells that are largely quiescent. Indeed, in in vivo studies in mice, intratumoral injection of as little as 104 infectious units of RCR vectors could spread and transmit a transgene throughout entire subcutaneous and intracranial tumor masses, whereas spread to normal tissues was undetectable.27Solly SK Trajcevski S Frisen C Holzer GW Nelson E Clerc B et al.Replicative retroviral vectors for cancer gene therapy.Cancer Gene Ther. 2003; 10: 30-39Crossref PubMed Scopus (66) Google Scholar, 32Wang WJ Tai CK Kasahara N Chen TC Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors.Hum Gene Ther. 2003; 14: 117-127Crossref PubMed Scopus (70) Google Scholar Second, they are inherently non-cytolytic and their cytolytic power hinges on the transfer of suicide genes followed by administration of a non-toxic pro-drug that is converted intracellularly to a cytotoxic substance by the suicide gene encoded enzyme. This provides both a means for control, as tumor cell killing depends on pro-drug administration, and for safety, as the same pro-drug could stop inadvertent vector spread. In in vivo studies in mice, intratumoral injection of RCR vector supernatant transducing a suicide gene, followed by pro-drug administration, resulted in 100% survival over a 100-day follow-up period, compared to 0% survival of control groups receiving either vector or pro-drug alone.32Wang WJ Tai CK Kasahara N Chen TC Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors.Hum Gene Ther. 2003; 14: 117-127Crossref PubMed Scopus (70) Google Scholar Furthermore, they achieved better therapeutic results than those reported using 10,000-fold higher levels of RDR vector supernatant.33Tamura K Tamura M Ikenaka K Yoshimatsu T Miyao Y Nanmoku K et al.Eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene.Gene Ther. 2001; 8: 215-222Crossref PubMed Scopus (18) Google Scholar It is thus clear that RCR vectors, as currently designed, are remarkably more efficient than RDR vectors to treat malignancies and, importantly, when administered to immunocompetent animals, their dissemination cannot be detected even by use of relatively stringent methods.32Wang WJ Tai CK Kasahara N Chen TC Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors.Hum Gene Ther. 2003; 14: 117-127Crossref PubMed Scopus (70) Google Scholar Therefore, they may be considered in settings with dismal prognosis and lack of alternative effective therapies (e.g., advanced malignancies such as recurrent glioblastoma multiforme). However, both RCR and RDR vectors still suffer from the risk of inadvertent spread, and any one of the following steps implicated in retroviral gene transfer can impose unwanted side effects: vector production, transgene insertion and expression, target cell or tissue manipulation, and host immune responses. Semi-replication-competent retroviral vectors. Aiming to make retroviral vectors worth considering in less extreme settings, we recently developed the so-called semi-replication-competent retroviral (s-RCR) vectors that are more efficient than RDR vectors and safer than RCR vectors. This alternative RCR vector design is based on two transcomplementing RDR vectors, each transducing a transgene and either gag-pol or env (Figure 2) that together, but not alone, hold all the genetic material necessary for replication and s-RCR vector production upon cell transfection (Figure 3). The s-RCR vector duo allows co-propagation of two different transgenes, which offers both a back-up therapeutic opportunity, should the effect of the first gene product wane owing to the development of drug resistance, and a means for vector replication shut off, if the transgene is a suicide gene. Furthermore, an s-RCR vector trio, i.e., mobilization of a third RDR vector including a third transgene, is also possible and further augments the system's versatility. With respect to efficacy, transgene propagation from s-RCR vectors was remarkably efficient both in vitro and in vivo, although not quite on par with RCR vectors.34Trajcevski S Solly SK Frisen C Trenado A Cosset FL Kaltzmann D Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors.J Gene Med. 2005; 7: 276-287Crossref PubMed Scopus (16) Google Scholar On the other hand, with respect to safety, the s-RCR vector did not lead to detectable dissemination or leukemogenesis, as did the RCR vector, after direct intravenous vector injection and subsequent bone marrow transplantation in MLV-sensitive mice.34Trajcevski S Solly SK Frisen C Trenado A Cosset FL Kaltzmann D Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors.J Gene Med. 2005; 7: 276-287Crossref PubMed Scopus (16) Google Scholar Beyond this approach, many others can be imagined. Most means currently explored to improve the integral safety of RDR vectors could be similarly applied to RCR vectors, including (i) physical targeting of cells of choice via modification of the envelope protein or expression from heterologous vectors,35Karavanas G Marin M Salmons B Gunzburg WH Piechaczyk M Cell targeting by murine retroviral vectors.Crit Rev Oncol Hematol. 1998; 28: 7-30Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 36Buchholz CJ Stitz J Cichutek K Retroviral cell targeting vectors.Curr Opin Mol Ther. 1999; 1: 613-621PubMed Google Scholar (ii) transcriptional targeting via replacement of the viral enhancer/promoter elements with tissue-specific regulatory sequences,37Zhao-Emonet JC Marodon G Pioche-Durieu C Cosset FL Klatzmann D T cell-specific expression from Mo-MLV retroviral vectors containing a CD4 mini-promoter/e