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Mol Ther. Mar 2010; 18(3): 477–490.
Published online Jan 19, 2010. doi:  10.1038/mt.2009.319
PMCID: PMC2839421

Recent Advances in Lentiviral Vector Development and Applications


Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution. They are also useful to generate induced pluripotent stem cells. LVs can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Targetable LVs can be generated by incorporating specific ligands or antibodies into the vector envelope. Immune responses toward the transgene products and transduced cells can be repressed using microRNA-regulated vectors. Though there are safety concerns regarding insertional mutagenesis, their integration profile seems more favorable than that of γ-retroviral vectors (γ-RVs). Moreover, it is possible to minimize this risk by modifying the vector design or by employing integration-deficient LVs. In conjunction with zinc-finger nuclease technology, LVs allow for site-specific gene correction or addition in predefined chromosomal loci. These recent advances underscore the improved safety and efficacy of LVs with important implications for clinical trials.


Lentiviral vectors (LVs) have become some of the most widely used vectors for fundamental biological research, functional genomics, and gene therapy. LV resembles γ-retroviral vectors (γ-RVs) in their ability to stably integrate into the target cell genome, resulting in persistent expression of the gene of interest. However, in contrast to γ-RV, LV can also transduce nondividing cells. This distinctive feature paves the way toward many applications for which γ-RVs are not suitable. Moreover, LV can accommodate larger transgenes [up to ~10 kilobases (kb)] compared to when γ-RVs are used,1 though vector titers tend to decrease with larger inserts.2,3 The focus of this review is to highlight some of the recent advances in LV technology and applications using HIV-1-based vectors. At the same token, only the salient features of some of its underlying basic vectorology will be presented because this was previously discussed.4,5,6,7,8

Vector Design and Production

Because HIV-1 is a human pathogen, it is critically important to ensure that the corresponding LV is replication-defective. The latest generation LV technology has several built-in safety features that minimize the risk of generating replication-competent wild-type human HIV-1 recombinants. Typically, LVs are generated by trans-complementation whereby packaging cells are co-transfected with a plasmid containing the vector genome and the packaging constructs that encode only the proteins essential for LV assembly and function5,9 (Figure 1). The self-inactivating (SIN) LV configuration reduces the likelihood that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed by abolishing the intrinsic promoter/enhancer activity of the HIV-1 LTR.10 The transcriptional inactivation of the LTR in the SIN provirus should also prevent mobilization by replication-competent virus upon subsequent infection with wild-type HIV-1. Consequently, this SIN feature minimizes the risk of horizontal and/or vertical LV transmission. Though the risk of vector mobilization is reduced with SIN LV, it is not totally eliminated.11 Several studies showed that even SIN vectors can give rise to full-length genomic transcripts competent for encapsidation and integration.12 An estimated 1/3,000 integrated vector genomes is transcribed, possibly due to de novo transcription from cryptic promoters either within or upstream of the integrated vector genome.13 LVs depend on reverse transcriptase to generate a transcription-competent double-stranded (ds)DNA template. The ability to convert the single-stranded (ss)RNA LV genome into dsDNA may be limiting in some cell types. For instance, LV transduction of human macrophages is relatively inefficient, possibly due to the limiting intracellular dNTP concentration that affects reverse transcriptase activity.14,15 Moreover, reverse transcriptase is relatively error-prone that may result in the emergence of mutations in the LV genome, including the transgene.

Figure 1
Lentiviral vector production by trans-complementation. Packaging cells are transfected with the lentiviral vector plasmid and three helper (i.e., packaging) constructs encoding Gag, Pol, Rev, and VSV-G. Only the vector contains the packaging sequence ...

The most common procedure to generate LVs is to co-transfect cell lines (e.g., 293T human embryonic kidney cells) with vector plasmid and the gag-pol, rev, and env packaging constructs. The Rev protein binds on the Rev responsive element that is required for the expression of the gag-pol transcripts by enhancing their extranuclear export. The typical titer of the nonconcentrated vector batches is about 107 TU (transducing units)/ml. This can be increased further to 109–1010 TU/ml by ultrafiltration or ultracentrifugation. Although transient transfection can produce high-titer LV, this method is cumbersome and difficult to scale up that poses significant manufacturing and regulatory hurdles. To overcome these limitations, stable packaging cell lines are being developed that already stably express the essential viral genes necessary to produce the viral vector particles. However, making stable packaging lines for LV production turned out to be more challenging than was initially anticipated.16,17,18 This could be ascribed mainly to the intrinsic cytotoxicity of the lentiviral protease encoded by the pol gene. In addition, the heterologous envelope protein [i.e., vesicular stomatitis virus G glycoprotein (VSV-G)] is also toxic when expressed in the packaging cells. To overcome these limitations, it is possible to drive the expression of the potentially cytotoxic proteins from an inducible promoter, provided expression is tightly controlled to avoid leaky expression of these cytotoxic proteins.19,20,21 Inducible stable packaging cell lines were recently developed that allow for large-scale LV production. In these systems, VSV-G and Rev expression is tightly regulated at the transcriptional level, using the Tet-On, Tet-Off, or cumate switch. These stable packaging cell lines allowed for the production of high-titer LVs (> 107 TU/ml) for several months, with no evidence of vector rearrangements. To generate high-titer SIN vectors, packaging cells were stably transfected by concatemeric array transfection. These new packaging cell lines may facilitate large-scale GMP-grade vector production. However, to our knowledge, they have not yet been used in clinical trials. Downstream processing typically involves additional vector purification or concentration steps that are typically based on ultracentrifugation, anion exchange chromatography, and gel filtration.22,23,24 Despite these recent improvements, LV titer remains relatively low compared to the titers that can typically be achieved with other viral vectors.

Gene Transfer Concepts and Potential Applications

Target cells and diseases

The ability of LV to transduce nondividing cells has prompted many different gene therapy applications. Moreover, the capacity of LV to accommodate relatively large transgenes is an advantage over adeno-associated viral vectors (AAVs), which have an intrinsic packaging limit of ~5 kb. Nevertheless, it is not always straightforward to compare the overall efficiency of LVs with that of other vector systems because different vector doses and expression cassettes are employed. This justifies the need for side-by-side comparisons under standardized conditions. Stable transduction was reported in different tissues with varying transduction efficiency depending on the target tissue. In particular, local administration of LVs into the brain or retina resulted in stable transduction of terminally differentiated cells.9,25,26,27,28,29 LVs have been demonstrated to transduce most cell types within the central nervous system (CNS) in vivo, including neurons, astrocytes, adult neuronal stem cells, oligodendrocytes, and glial cells.29,30,31,32 LVs have been used widely for gene transfer into the CNS and for gene therapy in neurologic disease models. After injection into the rat striatum, almost 90% of transduced cells were found to be neurons, with similar results found in rat hippocampus, primate striatum, and substantia nigra. However, this neuronal preference reflects the type of promoter used. If the promoter is active in glial cells, high-level transgene expression in astrocytes can be achieved. It is thus likely that LV enter many different cell types in the CNS with comparable efficiencies and that the different transduction patterns that have been reported are due to the activity of the internal promoter in the various cell types. Interestingly, depending on the envelope used to pseudotype the vector, some LVs can undergo retrograde transport in motor neurons, which has broad implications for gene therapy of motor neuron disease.33 One of the challenges of using LVs for gene transfer into the CNS is that they cannot readily cross the blood–brain barrier. Moreover, LV transduction remains essentially confined to the area near the injection site. In contrast, AAV vectors can lead to a more widespread transduction and, depending on the serotype, are capable of crossing the blood–brain barrier (i.e., AAV9), possibly via transcytosis.34,35

Sustained transgene expression could also be achieved by LV transduction of the skeletal muscle.36 However, LV transduction of skeletal muscle is not as efficient compared to when AAV vectors are employed, particularly those based on alternative AAV serotypes.35,37 As an alternative to direct intramuscular gene delivery, LVs can transduce different types of muscle stem cells that can differentiate into and/or fuse with muscle fibers in vivo. In particular, LVs are well suited to deliver therapeutic genes into mesoangioblasts that can functionally repair dystrophic muscle after systemic injection in dystrophic mouse and dog models.38,39 For these ex vivo applications, AAV vectors would not be suitable because most vector genomes do not integrate into the target cell chromosomes. Consequently, this would result in the concomitant loss of the nonintegrated therapeutic gene during cell division.

Direct intramyocardial delivery of LVs in adult rats bypasses the endothelial barrier and resulted in stable but overall modest transduction, confined primarily to the area near the injection site (<5%). Forced diffusion into the myocardium enhanced the overall LV transduction efficiency.40,41 However, none of these approaches resulted in the level of cardiac transduction efficiencies that could be attained with AAV, particularly AAV9.42,43,44 In adult mice, the heart was not permissive for LV transduction following systemic delivery. However, significant gene transfer could be detected in cardiomyocytes of neonatal recipients,45 possibly reflecting improved access.

Systemic administration typically resulted in widespread transduction of hepatocytes and antigen-presenting cells (APCs), including Kupffer cells and splenic APCs.36,45,46,47 Whereas γ-RVs cannot transduce nondividing hepatocytes, LVs could overcome this limitation. Hepatic transduction efficiency with LVs compared favorably with that obtained with AAV2 vectors. However, AAV2 requires portal vein injection, whereas LVs can achieve comparable levels of gene transfer by systemic administration. We recently demonstrated the superior hepatic transduction efficiency with AAV8 or AAV9 vectors compared to LVs, at least in mice.42 However, this may not necessarily translate to increased efficiencies in large animal models or ultimately in human subjects. Ultimately, the choice of vector for hepatic gene delivery may thus not only depend on their relative efficiencies but also on their respective adaptive and innate immune reactions (see below). Future translational studies in large animals are therefore needed to resolve this outstanding issue.

The ability to transduce these different tissues with LVs opens up new possibilities for gene therapy of hereditary, acquired, or complex disorders that affect these various organs (e.g., neurodegenerative diseases including adrenoleukodystrophy (ALD), metachromatic leukodystrophy, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, congenital blindness, liver disease, hemophilia, metabolic diseases, and lysosomal storage disorders, etc.).28,29,30,31,32,33,42,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62 Proof of concept has been established that LVs can result in effective gene therapy for these various diseases in preclinical animal models that mimic the cognate human disease. LVs are also suitable for the transduction of various stem/progenitor cells, particularly including hematopoietic stem cells (HSCs).63,64 Stable expression of the transgenes at therapeutically relevant levels has been demonstrated following LV transduction of HSC for a variety of hematologic and metabolic diseases (e.g., hemoglobinopathies, severe combined immune deficiencies, hemophilia, etc.).2,53,65,66,67 However, for some of these applications, a high copy number was required to achieve these expression levels and some clones contained >5 vector copies per cell. Obviously, such elevated copy numbers preclude clinical translation because it is associated with an increased risk of insertional oncogenesis (see below). It is therefore important to continue to refine the LV design and/or transduction conditions to the extent that therapeutically relevant levels of the transgene product can be achieved while maintaining low vector copies per cell values. This may warrant the use of more potent lineage and/or tissue-specific promoters to drive the transgene in the context of SIN LV backbones equipped with insulator sequences (see below).

Though ex vivo gene therapy is the most commonly used approach to transduce HSC, direct in situ LV transduction is a potentially attractive alternative and may overcome some of the intrinsic limitations of ex vivo gene delivery. In particular, the in situ approach avoids ex vivo culture of target cells.68 Ex vivo culture of HSC, progenitors, and even mature T cells results in impaired homing capabilities and functional deficits. Furthermore, ex vivo culture potentially exposes the cells to infectious agents, serum, and other risks related to complex and prolonged manipulations and removes them from their natural microenvironment. Relatively efficient gene marking was observed following direct intrafemoral injection of an LV in mice 4 months after injection yielding up to 12% green fluorescent protein–expressing cells in myeloid and lymphoid sub-populations.69 Most importantly, this resulted in almost 10% transduction efficiencies in HSC and relatively efficient gene marking in secondary bone marrow transplants. Integration analysis confirmed that multiple transduced clones contributed to hematopoiesis. LVs have also recently been explored to achieve in situ transduction of T-cell precursors by direct injection into the thymus.70 In particular, partial reconstitution of polyclonal and functional T cells could be achieved by direct injection of a LV encoding human ZAP-70 into ZAP-70–/– mice. This approach avoids transduction of HSC and precursor cells from other hematopoietic lineages that do not express ZAP-70 and thus do not require correction in this disorder. This potentially reduces the risk of insertional mutagenesis by exposing fewer precursor cells to insertional events. HSC may be particularly susceptible to insertional mutagenesis due to their prolonged and increased replicative capacity. Many genes controlling self-renewal and proliferation are expressed in HSC before being shut off during differentiation, and increased expression of genes has been shown to predispose loci to insertions of γ-RVs and LVs.71 Insertions at these sites could therefore constitutively activate genes that are normally shut off during hematopoietic development, and thus contribute to leukemogenesis. By targeting more mature cells via intrathymic injection, this risk might be decreased. However, more long-term studies are needed to assess the long-term efficacy and safety of this approach.

Gene regulation

LVs have been generated containing lineage-specific promoters that upon transduction of HSC are only expressed in the desired lineage upon hematopoietic reconstitution. For instance, the megakaryocytic GpIIb promoter and the erythroid-specific globin promoter resulted in specific expression of the gene of interest in the corresponding hematopoietic lineages.72,73,74 The ability to achieve erythroid-specific expression using globin-specific regulatory elements in the context of LVs was critically important to achieve robust and stable therapeutic effects in globinopathy animal models.2,75,76 These preclinical advances paved the way toward a phase 1 gene therapy clinical trial for β-thalassemia major.76 The use of lineage-specific regulatory elements prevents ectopic expression of the gene of interest in other cell types and improves the overall efficacy and safety of the HSC-directed LV approaches. Moreover, the use of promoter/enhancers that are only active in terminally differentiated, nondividing cells may reduce the risk of insertional oncogenesis (see below). In particular, coaxing expression of the gene of interest to the erythroid and megakaryocytic/platelet lineages is particularly appealing from the safety perspective because erythrocytes and platelets do not contain a nucleus. However, some of these erythroid or megakaryocytic promoters are still active in their cognate progenitors that contain a nucleus. Consequently, the occurrence of insertional oncogenic events in these progenitors cannot be excluded, which may ultimately lead to their malignant transformation. Nevertheless, these lineages provide an attractive platform to deliver secretable proteins directly into the circulation. In particular, it has been shown that transduction of HSC with LV can result in erythroid-specific or platelet-specific expression of clotting factors that consequently correct the bleeding diathesis in hemophilic mice.77 This is an attractive gene therapy approach for hemophilia that needs to be validated further in large animal models. It is likely that in vivo selection would ultimately be required to enrich for the gene-modified cells in addition to the need for myeloablative conditioning.78 Interestingly, localized production of clotting factors inside the platelet plug protects the clotting factor from inactivation by neutralizing antibodies.72,79 The ability to obtain robust lineage-specific expression is an attractive paradigm to control the expression of the gene of interest in the appropriate cell type and has currently been explored for HSC-based gene therapy for Wiskott–Aldrich and other hematopoietic and lysosomal storage disorders, including Hurler syndrome.80,81,82,83 Finally, LV-mediated gene transfer into HSC has proven to be effective for the treatment of neurodegenerative disease including metachromatic leukodystrophy and ALD by targeting expression of the therapeutic protein into the microglial compartment.58,59,84 Alternatively, drug-inducible systems can be used in conjunction with LVs to better control the levels and/or timing of gene expression85 (e.g., tetracycline, ecdysone, and mifepristone-inducible systems.86,87 However, there are some concerns about the immunogenicity of the various transactivators used in these regulatable systems that may curtail long-term persistence of the genetically modified cells.

In addition to the use of lineage-specific promoters, it is also possible to obtain lineage-specific expression using a novel paradigm based on microRNA (miR) regulation.88,89,90 To achieve this, a miR target sequence is incorporated in an LV that expresses the gene of interest. This miR target sequence is complementary to an endogenous miR that is specifically expressed in a given lineage. The specific interaction of this lineage-specific miR with its cognate miR target sequence embedded within the vector mRNA results in its degradation via the cellular miR processing machinery. Consequently, this prevents expression of the gene of interest in a lineage-specific fashion and hereby enhances the versatility of tissue and/or cell type–specific LVs. LVs have also recently been employed to stably and specifically knock down miR by overexpressing miR target sequences, that consequently act like molecular sponges or decoys for the endogenous complementary miR. This provides an attractive platform technology to repress the function of endogenous cellular miR. Nevertheless, high copy number and transduction efficiencies were sometimes required to effectively repress miR expression. Alternatively, LV can be used to overexpress miR sequences rather than their cognate targets. Some miRs also provide a favorable scaffold for expression and optimal processing of shRNA.91 When incorporated into LV, these shRNA molecules are capable of inhibiting mRNA by RNA interference and therefore provide an attractive paradigm for inhibition of viral replication92 or for the treatment of dominant genetic diseases (e.g., Huntington's disease) by gene therapy.93


VSV-G is the most commonly used viral envelope protein used for LV pseudotyping, but other envelopes including rabies, murine leukemia virus-amphotropic, Ebola, baculovirus, and measles virus (MV) envelopes have also been used94 (Figure 1). Pseudotyping HIV-1 vectors obviate safety concerns associated with the use of HIV-1 gp120, which has known pathogenic consequences. Moreover, pseudotyping has a dramatic impact on the biodistribution and vector tropism. In particular, HIV-1 gp120 restricts transduction of HIV-1 vectors to CD4+ cells that limits its usefulness for gene therapy applications to CD4+ cells, like T cells or macrophages. In contrast, heterologous envelopes, like VSV-G, typically broaden the tropism and allow gene transfer into a broad variety of cells in vitro (e.g., CD34+ stem cells) and in vivo (e.g., brain, muscle, and liver). The choice of envelope used to pseudotype the LV is at least partly determined by the target cell or tissue that needs to be transduced. For instance, filovirus envelope-pseudotyped LV enhance transduction of airway epithelia or endothelial cells,95 whereas baculovirus GP64 and hepatitis C E1 and E2 pseudotyping enhances hepatic transduction.96,97 The choice of envelope also seemed to have an impact on the tropism and trafficking of LVs in the CNS. In particular, pseudotyping LV with rabies virus glycoprotein enables retrograde axonal transport and access to the nervous system after peripheral delivery.98,99,100 RD114 pseudotyping favors transduction in lymphohematopoietic cells. LVs pseudotyped with the Edmonston MV glycoproteins H and F allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells.101 Indeed, these H/F-displaying vectors outperformed VSV-G pseudotyped LVs for the transduction of IL-7-prestimulated and quiescent T cells. In resting lymphocytes, postentry steps of HIV infection, and possibly also of LV transduction, such as completion of reverse transcription, nuclear import, and chromosomal integration of the transgene, do not occur.102 This blockade may be due to the presence of a dense cortical actin layer in quiescent T cells impeding transfer of the viral or vector genome from the cellular periphery to the microtubule network. This ultimately results in a reduction of reverse transcription and nuclear import.102,103,104 The MV pseudotyped LVs may potentially overcome this bottleneck by inducing rearrangement of the actin network and allowing the vector core to be transported to the nucleus in the absence of a proliferative stimulus. This hypothesis is consistent with the observation that certain strains of HIV and MV trigger actin rearrangements in resting lymphocytes upon contact with their cell surface.103,105 The interaction of HIV with its native CXCR4 co-receptor results in the activation of cofilin that subsequently causes the dissolution of the actin cortical blockade and enhanced transport of the viral particles to the nucleus. Similarly, the interaction of MV with SLAM and/or CD46 activates cofilin and moesin resulting in dissolution of the actin barrier and enhanced formation of stable microtubules.

Cell type–specific targeting

Cell type–specific LV can be generated by incorporating cell type–specific ligands or antibodies into the viral envelope (Figure 2). Modification of the envelope with these cell type–specific retargeting moieties can redirect the binding of the LV particles to the corresponding cellular receptor. However, envelope engineering can potentially alter the fusion domain of Env resulting in low vector titers. It is therefore important to ensure that redirecting vector tropism does not compromise the postbinding steps in vector entry. To address this concern, fusogenic envelope proteins are incorporated alongside the retargeted envelope proteins into the LV particles. Typically, the fusogen is constructed by modifying viral envelope proteins to the extent that they lack the ability to bind to their cognate receptor but still retain the ability to trigger membrane fusion. Thus, the specificity of such an LV is then solely determined by the retargeting moiety.

Figure 2
LV targeting into specific cell types. The LV Env protein is amenable to engineering allowing the display of cell type–specific ligands or single chain antibody fragment (scFv). These ligands or scFv can then bind onto cell surface receptors that ...

To establish proof of concept, LV had been generated that display a covalently linked mouse/human chimeric CD20-specific antibody on their surface. This retargeted LV recognizes and binds to the corresponding CD20 antigen on the target cell membrane.106,107 Antibody binding subsequently induces endocytosis, bringing the LV into the endosomes where the fusogenic molecule (i.e., Sindbis virus envelope) responds to the low pH environment and triggers membrane fusion. This allows the LV core to enter the cytosol. Ultimately, this retargeting principle resulted in specific and stable transduction of CD20+ human lymphoid B cells. Moreover, retargeted LV equipped with a suicide gene caused in vivo suppression of xenografted tumors expressing CD20. These results show the feasibility of retargeted LV for therapeutic applications.106 CD20-retargeting may activate the resting B cells allowing transit from the G0 to G1 phase of the cell cycle, consistent with increased intracellular Ca2+ levels.102 The main advantage of this method is that it can be extended to other antibodies and cell surface receptor–ligand pairs107 (Figure 2). Recently, this LV retargeting paradigm has been further optimized using either scFv or ligands (e.g., epidermal growth factor) genetically fused to an MV hemagglutinin envelope. This modified HA does not recognize its native receptor. Direct fusion at the cell membrane is mediated by the MV F envelope in a pH-independent manner, which does not need to be internalized in contrast to Sindbis envelopes.108 Though LVs can transduce nonproliferating cells, transduction efficiency in resting T cells and HSCs is not highly efficient. By engineering LV particles displaying T-cell-activating single chain antibody peptides, resting T cells could be transduced.109 Alternatively, LVs displaying IL-7 increase transduction of resting T cells without inducing a naive-to-memory phenotypic switch.110 Similarly, to obtain more selective and efficient gene transfer into HSC, recombinant membrane proteins were engineered and were incorporated into LV particles to display “early-acting” cytokines on their surface. To achieve this, a recombinant membrane protein consisting of the transmembrane influenza hemagglutinin glycoprotein was fused to truncated forms of thrombopoietin. Similarly, the stem cell factor (SCF) was fused to the amphotropic Moloney murine leukemia virus env glycoprotein. LVs displaying SCF and thrombopoietin can bind their cognate cellular receptors on HSC (i.e., c-mpl and c-kit). In addition, these LVs bind the receptors for VSV-G, which are phospholipids in the HSC membrane. With these engineered vectors displaying thrombopoietin, SCF, or both cytokines, transduction efficiency of quiescent CD34+ cells was significantly increased. These surface-engineered LVs preferentially transduced and promoted survival of resting CD34+ cells rather than cycling cells. Importantly, these novel LVs allowed superior gene transfer into the most immature CD34+ cells as compared with conventional LVs, even in the presence of cytokines. LV displaying these “early-acting cytokines” were able to selectively transduce HSC with high efficacy and without disturbing their function or differentiation potential.111 The use of SCF-retargeting in conjunction with a mutated RD114 envelope resulted in a further enhancement of CD34+ HSC transduction compared to when VSV-G was used as fusogen112 (Figure 2). These novel LVs even allowed for relatively robust in vivo targeting to CD34+ HSC that were transplanted into immunodeficient mice113 paving the way toward a clinically relevant stem cell targeting paradigm. The interaction between SCF and its cognate cellular receptor c-kit may not merely serve as a targeting modality sensu stricto, but may potentially trigger signaling events that facilitate the subsequent steps of lentiviral transduction, reminiscent of what has been reported for MV and HIV.102,103,104,105 It would be particularly interesting to find out whether the SCF/c-kit interaction influences cytoskeletal remodeling by regulating actin rearrangements and microtubule formation enabling transport of LV particles to the nucleus.102,103,104 It is not clear whether the particles that stimulate the c-kit receptor are also those that enter the cell.


Because a functional integrase is required to achieve stable genomic lentiviral integration, it is possible to prevent this integration by mutational inactivation of the integrase protein. The use of integration-defective LV (IDLV) could potentially obviate concerns associated with random integration and insertional oncogenesis (see below).114,115,116 However, it is important to ensure that the mutational inactivation only affects the integration process per se without compromising other steps in the transduction process. Therefore, the development of clinically relevant IDLV should be based upon class I integrase mutations that result in a specific defect in integration, as opposed to class II mutants that exhibit additional assembly or reverse transcription defects that consequently affect integration. The quintessential class I mutation D64V leads to a robust decline in genomic integration and has been adopted for use in IDLV. Consequently, these IDLVs have a defect in integration characterized by accumulation of double-stranded episomal DNA circles in the host cell nucleus. These episomes can either be 1-LTR circles, that arise following intramolecular homologous recombination between the LTRs, or 2-LTR circles that are formed as a result of intramolecular end joining of the two LTR ends. Though it was initially assumed that IDLV would be unable to support robust transcription of the gene of interest,9 later studies demonstrated that IDLVs are transcription-competent and can result in relatively efficient expression of the gene of interest.114 In dividing cell populations, transgene expression from IDLV is initially comparable to that of integration-competent LVs. However, expression subsequently declines due to the loss of the nonintegrated IDLV episomes, as the cells progress through subsequent cell cycles. Therefore, IDLVs can be used to achieve short-term expression of a gene of interest in dividing cells. This property of IDLVs is exploited to transiently express zinc-finger nuclease (ZFN) in the context of gene targeting (see below). However, in nondividing target cells, in particular retina, brain, liver, or skeletal muscle, transgene expression from IDLV is relatively stable.117,118,119,120 In retina and brain, IDLV seems to be at least as efficient as bona fide integrating LV at both high and lower vector doses. This resulted in functional rescue of congenital blindness in a rodent model of autosomal recessive retinitis pigmentosa resulting from a mutation in rpe65.114 The stable transgene expression from IDLVs is consistent with the persistence of the lentiviral circular episomes. IDLV is also becoming an attractive platform technology for genetic vaccination. In particular, muscular gene delivery or subcutaneous administration of IDLVs resulted in transduction of dendritic cells and antigen presentation for at least a month that consequently resulted in the induction of immune responses to viral antigens.121,122 However, efficiency of IDLVs may vary depending on the target tissue. In particular, transduction of hepatocytes in mice is less efficient with IDLVs than when integrating LVs are employed (ref. 120 and T. VandenDriessche, unpublished results). This may reflect—at least in part—the intrinsic challenge of achieving high multiplicities of infection in the liver compared to the retina or brain.

Though genomic integration of IDLVs is clearly impaired, the D64V mutation in the integrase does not totally abrogate integration. Consequently, these residual integrated vectors can contribute to insertional mutagenesis and may express the gene of interest. Expression would persist in a dividing target cell population, particularly if the gene of interest confers a selective pressure to the transduced cells. To further reduce the emergence of residual integration, it may be warranted to include multiple mutations in the integrase and/or the att site in the LTR that is required for integration. However, due to intrinsic cellular recombination events, low-frequency residual integrations cannot be excluded. IDLVs have residual integration frequencies 3- to 4-log below those of the cognate integration-competent LV, at least in cell culture114 and are within the range described for plasmid transfection. Genomic integrations from IDLV typically reflect noncanonical, integrase-independent events (ref. 117 and T. VandenDriessche, unpublished results). Consequently, though the risk of insertional mutagenesis from IDLVs is highly reduced compared to that from integrating LV, even this minor level of residual integration carries an intrinsic genotoxic risk. However, more exhaustive integration site analyses are needed to assess the integration profile of these noncanonical IDLV integrants.

Hybrid technologies

LV/transposon hybrid. The Sleeping Beauty (SB) transposon system is an attractive gene delivery platform allowing stably integration of the gene of the interest through transposition into the target cell genome.123,124 This transposition requires the interaction between the SB transposon inverted repeats and the SB transposase, that is typically encoded in trans. Though the stable gene transfer efficiency with the early-generation SB transposases in most primary cells was relatively poor, we have recently characterized hyperactive SB transposases that were obtained by Darwinian evolution and selection in vitro that yield increased stable gene delivery efficiencies in clinically relevant primary cells, including HSC.123 SB does not exhibit a preference for integration within active genes. Moreover, the SB inverted repeats have only very low residual promoter/enhancer activity. These features improve the safety profile of SB transposons compared to LVs or γ-RVs by reducing the risk of insertional oncogenesis. To combine these favorable attributes of SB transposons with the efficient gene delivery properties of LV, hybrid vectors were constructed in which the SB transposon and SB transposase are encoded by IDLV.125,126 IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats but devoid of HIV-1 LTRs or other virus-derived sequences. Most importantly, integration site analysis revealed redirection toward a profile mimicking SB integration and away from integration within transcriptionally active genes favored by integrating LV. However, the overall efficiency of this hybrid technology, even in conjunction with the use of the hyperactive SB100X transposase,126 is still not comparable to that of bona fide LV, nor has it been validated in clinically relevant target cells. Finally, residual integration of IDLVs may result in low-level continuous transposase expression that could result in transposon hopping and mobilization.

LV/ZFN hybrid. Gene correction by homologous recombination is a promising gene therapy modality for the treatment of monogenic diseases. It allows for in situ correction of a defective gene. Gene correction has several advantages: (i) it avoids potential risks associated with (quasi)random genomic integration and in particular insertional oncogenesis, and (ii) it preserves the natural expression pattern of the corrected gene and consequently avoids potential complications associated with ectopic expression of the gene of interest. In general, the overall efficiency of homologous recombination is extremely low that hampers clinical applications. However, it is possible to overcome this limitation by introducing double-stranded DNA breaks (DSB) in the desired target gene that needs to be corrected that results in a dramatic increase in efficiency of homologous recombination with several orders of magnitude. Introducing a DSB into the target DNA increases the interaction between the host and an exogenous homologous DNA sequence by mobilizing the homologous recombination repair mechanism.127 Engineered ZFNs are attractive molecular tools to achieve more precise and efficient gene correction at the targeted locus (Figure 3). Typically, ZFNs are modular and correspond to a C2H2 class of zinc-finger DNA-binding domain, specifically engineered to recognize the DNA sequence of interest, and fused to the nuclease domain of the FokI endonuclease. By virtue of the specificity of the designer zinc-finger proteins, the FokI endonuclease is then “recruited” to the target locus where it catalyzes the DSB. This subsequently allows the exogenous homologous DNA to correct the cognate defective sequence. Because FokI must dimerize to be able to induce DSBs, two different ZFNs, which bind the DNA at palindromic sequences of the complementary DNA strands, need to be delivered into the cells of interest along with the exogenous DNA sequence. This co-delivery of ZFNs and exogenous DNA can be accomplished using IDLVs128 (Figure 3). Proof of concept has been established that IDLV-ZFN vectors can achieve relatively efficient gene correction at a desired locus. However, the efficiency of gene correction is depending on several confounding variables, including the targeted locus, the transduction efficiency, and the target cell type. Typically, higher gene correction efficiencies can be achieved in cell lines compared to primary cell types and may be related, at least partly, to their difference in proliferation status. To achieve successful gene correction, proximity of the mutation to the ZFN cleavage site is necessary. This implies that each mutated locus of interest would require ZFNs tailored to that site. However, using the same principles, it is also possible to use ZFNs to “knock in” corrected genes into the cognate genes that carry a broad spectrum of mutations, obviating the need to make a different ZFN for each patient. Finally, using this IDLV-ZFN technology, it is possible to achieve targeted delivery of a gene of interest into a specific “safe harbor” chromosomal locus in which integration is believed to be safe (i.e., non-coding DNA), which would minimize the risks associated with random integration. Though promising, the technology warrants further optimization in terms of efficiency and safety. One key issue that needs to be addressed further is the potential “off-target” effect, whereby DSBs are introduced in other loci besides the desired locus and that may contribute to ZFN cytotoxicity.129 The underlying principles and parameters that influence the rate of “off-target” effects versus the intended targeted gene corrections are currently being investigated. Moreover, the IDLV delivery platform may still result in residual genomic integration (see above) that may result in inadvertent stable expression of ZFN or the exogenous DNA. Nevertheless, the use of ZFN technology in conjunction with IDLV is promising and is currently being explored for gene therapy of a variety of diseases. Recently, it has been shown that the gene encoding the HIV-1 co-receptor CCR5 can be “knocked out” in T cells using IDLV-ZFN vectors that render the cells relatively resistant to HIV-1 infection.130 This proof-of-concept study may ultimately pave the way toward a gene therapy clinical trial in HIV-1-infected patients.

Figure 3
LV chromosomal targeting. (a) A pair of integration-defective LVs vectors (IDLVs) each expressing ZFN are co-transduced with an IDLV providing the exogenous donor DNA template. (b) Gene correction is catalyzed by ZFN-mediated double-stranded DNA break ...

Immune Consequences

LV can trigger innate and adaptive immune reactions directed against the vector particles, the transduced cells, and/or the transgene product encoded by the vector.131 The immune response induced by LVs is a double-edged sword. It may contribute to more robust immune reactions, which would be a possible advantage in the context of genetic vaccination strategies. However, untoward immune responses may curtail expression of the gene of interest that would be undesirable when treating hereditary diseases that warrant stable transgene expression. The vector particles themselves can trigger a rapid but self-limiting proinflammatory response leading to a transient cytokine surge (e.g., interleukin-6)42 and IFNαβ response.132 This proinflammatory immune response can likely be ascribed to efficient interaction of the LV particles with APCs,45 and it has been proposed that this may involve engagement of toll-like receptor 7 (TLR7), a pattern-recognition receptor for single-stranded RNA (ssRNA) and/or TLR9 that recognizes unmethylated CpG. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNβ, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. This innate immune response seems less robust compared to when adenoviral vectors are employed. Nevertheless, it may still influence the adaptive immune response to the vector, the transgene product, and/or the LV-transduced cells. Exposure to LVs will likely induce vector-specific antibodies that will neutralize the vector particles and consequently prevent gene transfer by subsequent vector readministration. It is also possible, in principle, that pre-existing antibodies to the heterologous Env protein used to pseudotype the LVs may interfere with viral transduction. The risk of having pre-existing antibodies to the Env protein may depend on the virus from which the Env was derived and its prevalence in the human population. However, by carefully selecting the type of envelope used for pseudotyping, this risk can be significantly reduced. The use of LVs may therefore potentially overcome some limitations associated with the use of AAV vectors.133,134 In particular, it is known that pre-existing immunity to AAV capsids following natural exposure to AAV may induce cytotoxic T-cell (CTL) responses that could possibly eliminate transduced myofibers or hepatocytes, and curtail long-term gene expression.133,135 Because most human subjects have not been pre-exposed to LV components, this is likely to be less of a concern with LVs than with AAV.

The immune system of many patients suffering from hereditary diseases has not been tolerized to the functional transgene product that they are missing. Consequently, it is possible that LV transduction may evoke antigen-specific humoral and cellular immune reactions that could result in the elimination and/or neutralization of the transgene product and/or the clearance of the transduced cells. The induction of an antigen-specific antibody response against a secretable transgene product encoded by the LVs typically requires antigen processing via the exogenous pathway by APCs resulting in presentation of antigenic peptide in association with major histocompatibility complex class II.51,136,137 This, in turn, triggers a T-helper response that ultimately activates B cells to produce antibodies specific to the transgene product encoded by the LV. In addition, gene transfer may result in the presentation of endogenously synthesized peptides derived from the transgene product in the context of major histocompatibility complex class I molecules potentially resulting in cytotoxic T-cell responses (CTL) that could consequently eliminate the LV-transduced target cells. The magnitude of these antigen-specific humoral and cellular adaptive immune reactions following LV administration depends on several parameters, including the transgene product, vector design, vector dose, route of vector administration, target cell type, and genotype of the recipient animal or patient. In particular, LV transduction of APCs may result in ectopic expression of the transgene product. This may increase the risk of inducing humoral and/or cellular immune response that curtail long-term gene expression and particularly if LV transduction enhances the maturation of APC. To minimize this risk, it is warranted to use cell type–specific promoter/enhancers to restrict transgene expression to the target tissue while preventing inadvertent ectopic transgene expression in APCs.51,137 Nevertheless, even with highly tissue-specific promoters, immune response against the transgene product cannot always be prevented due to “leaky” transgene expression in APCs. To ensure robust tissue-specific expression of the transgene product and prevent ectopic expression in APC, an additional layer of regulation was built into the LV by incorporating a target sequence for the hematopoietic-specific microRNA, miR-142-3p (Figure 4). This eliminated off-target expression in hematopoietic cells, particularly APC, allowing for possible induction of immune tolerance and resulting in sustained expression of the gene of interest, in this case, FIX in a hemophilia B mouse model.88,137 This concept would need to be further validated in large animal models. Moreover, it is important to ensure that the miR target sequence does not compromise the normal function of its cognate complementary endogenous miR sequence. This potential risk may depend on qualitative and quantitative variables, such as the type of miR and the relative expression levels of a given miR versus its cognate miR target. Indeed, though forced overexpression of miR-142-3p target does not seem to downregulate or titrate the endogenous miR-142-3p, other miR targets can repress their cognate endogenous miR.93 However, miR-regulation may not necessarily suffice to induce immune tolerance in all circumstances, and additional studies are required to address these outstanding issues. In particular, even if ectopic transgene expression in APC is eliminated, it is still possible that immune responses to the transgene product can be induced.138 Indeed, the efficiency of immune tolerance induction by miR-regulation may vary depending on the transgene product, the target organ, and the underlying mutation of the defective endogenous gene. In particular, it is typically more challenging to induce immune tolerance in the context of a null mutation than when a missense mutation occurred in the affected gene. In any case, though miR-regulation may impact on the adaptive immune response against the transduced cells and/or the therapeutic proteins, it will not eliminate the innate immune response induced by the LV particles themselves.

Figure 4
micro-RNA regulated LV. Ectopic transgene expression in APC results in the induction of an antigen-specific T-cell dependent immune response that consequently eliminates the gene-engineered APC and hepatocytes that express the transgene product (i.e. ...

It is generally assumed that ex vivo gene delivery will obviate immune concerns related to direct exposure to LV particles. However, recent studies have shown that LVs can adhere to hematopoietic target cells leading to inadvertent secondary transduction and prolonged gene expression in recipient tissues.139 Intriguingly, LV attachment to the cell surface selectively protects the vector particles from serum complement-mediated inactivation allowing their subsequent in vivo dissemination. This has implications for LV biosafety and immunotoxicology.

Generation of iPS Cells

Induced pluripotent stem (iPS) cells are promising adult stem cells for regenerative medicine. iPS cells are derived from autologous somatic cells following genetic reprogramming and have first been described by Takahashi and colleagues.140,141,142 Typically, genetic reprogramming of mouse and human fibroblasts can be achieved following ectopic expression of a defined combination of four transcription factors, namely c-Myc, Klf4, Oct4, and Sox2.140,143,144,145,146,147,148,149 Other cell types already a priori express some of these factors, and consequently only require a minimal set of exogenously introduced reprogramming factors.150 The main advantage of iPS cells is their remarkable pluripotency, which resembles that of embryonic stem cells. iPS cells can be genetically modified and can be induced to differentiate into endodermal, mesodermal, and ectodermal cell types for transplantation to treat degenerative and/or genetic diseases. iPS cells can be obtained from autologous, histocompatible adult somatic cells, obviating the need for prolonged immunosuppressive therapy in the context of cell transplantation. In one landmark study, iPS cells derived from fibroblasts of sickle cell anemia mice were genetically corrected by replacing the mutant β-globin allele with a wild-type allele by means of homologous recombination. This provided a source of iPS cells able to differentiate into disease-free hematopoietic precursors that cured the afflicted mice following transplantation.151

Most prior studies have required multiple retroviral vectors for genetic reprogramming. This resulted in high numbers of genomic integrations in iPS cells and consequently precludes their use for therapeutic applications. LVs have recently been generated expressing a “reprogramming cassette” comprising four transcription factors. Relatively efficient reprogramming can be achieved by combining all factors into a single transcript. Moreover, this allows derivation of iPS cells with a single viral integration.152 One specific advantage of this “all-in-one” approach is that only those LV-induced iPS clones that contain a single LV integration in a “safe” chromosomal location can be preselected for further studies, which would minimize concerns of insertional oncogenesis (see below). However, a specific risk associated with the use of γ-RVs and LVs for iPS generation relates to the possible ectopic expression of the delivered transcription factors in the progeny of the reprogrammed cells. Because the c-Myc and Klf4 reprogramming factors are known oncogenes, their expression or reactivation in iPS-derived mice may cause tumors. To prevent (re)expression of the reprogramming factors in the iPS progeny, LVs have been generated in which the reprogramming cassette is flanked by lox P sites allowing subsequent excision upon de novo expression of the CRE recombinase in the iPS-derived cells.153 The use of a single LV for reprogramming in conjunction with a CRE-lox-based reversible genetic modification paradigm represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes. However, even upon CRE-mediated excision of the reprogramming cassette, a genetic trace of the integrated LV remains present in the iPS cells and their progeny. Only those clones in which this genetic remnant is present in an innocuous location in the genome will qualify for possible clinical applications. Nevertheless, further studies are required to assess whether LV-based reprogramming offers any advantages in terms of safety and efficacy over competing technologies that allow for “traceless excision” of the reprogramming cassette using PiggyBac-derived transposons,124,154 or that rely on protein and/or RNA-based reprogramming.

Safety Issues

The use of SIN vector configuration, heterologous envelopes, and Tat-independent vector production has significantly improved the overall LV safety. Moreover, potential homologous overlap between vector and packaging constructs is minimized to reduce the risk of generating replication-competent lentiviruses by homologous recombination. It is encouraging that replication-competent lentiviruses have not been detected in large-scale LV production batches based on the latest generation LV packaging system and vector designs, which further underscores their relative safety. However, some animals, such as wild-type mice, cannot support replication of infectious HIV-1. Consequently, it cannot be excluded that the potential for shedding of replication-competent lentiviruses from such animals may be underestimated, even if replication-competent lentiviruses were present in the original vector inoculum.

Though genomic integration is essential to obtain stable expression of the gene of interest following lentiviral transduction of dividing cells (e.g., following hematopoietic reconstitution), it is a double-edged sword that may contribute to genotoxicity reminiscent of the properties of γ-RVs. Indeed, it is now well established that the integration of a γ-RV encoding the IL2Rγc gene in proximity of the LMO2 proto-oncogene contributed to deregulated LMO2 expression and ultimately to leukemogenesis observed in several subjects enrolled in a clinical trial for SCID-X1.155,156 One group has suggested that ectopic expression of murine IL2Rγc under control of LV may contribute leukemogenesis because LV-mediated IL2Rγc gene transfer into HSC was associated with increased malignancy in hematopoietic reconstitution experiments in mouse models.157 In contrast, ectopic expression of human IL2Rγc in human hematopoietic cells investigated in T-cell differentiation assays in vitro or in murine bone marrow transplantation assays performed with long-term follow-up did not induce cell transformation per se.158,159 The risk of leukemogenesis prompted several investigations into the safety consequences of using integrating vectors for gene therapy. Consequently, one of the main potential safety concerns related to the use of LVs in clinical applications relates to its intrinsic ability to integrate into the target cell genome and to a much lesser extent to the HIV-1 pathogenicity of the wild-type virus from which they are derived. The intrinsic genotoxicity of γ-RVs versus LVs is being compared in several preclinical models to further address this safety issue. In general, LV or γ-RV exhibit semirandom insertion profiles with varying degrees of preference for actively described genes and distinct cis-regulatory regions such as CpG islands and DNAse 1 hypersensitive sites.160,161 Both LVs and γ-RVs show an integration bias into transcriptional units indicating that integration is not random per se. Nevertheless, the integration pattern of γ-RVs seem to differ from that of LVs. γ-RVs have a predilection toward integrating in the immediate proximity of transcription start sites and a small window around DNAse I hypersensitive sites, whereas LVs are more likely to integrate further away from the transcription start sites into active transcription units.3,129 To assess the relative oncogenicity/genotoxicity of LV, HSC gene transfer studies are being conducted in tumor-prone mouse models (i.e., Cdkn2a−/− mice).162 These studies uncovered low genotoxicity of LV integration compared to when γ-RVs were used. γ-RV triggered dose-dependent acceleration of tumor onset contingent on LTR activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by the SIN LVs and did not result in enrichment for specific integrants, despite the higher integration load and robust expression of LV in all hematopoietic lineages. Therefore, the prototypical LV appeared to have low oncogenic potential. However, in that study, a SIN LV design was compared with a non-SIN γ-RV. Comparisons of the intrinsic genotoxicity/oncogenicity of SIN LV with SIN retroviral vectors would be preferred because recent studies clearly indicate that SIN γ-RVs have a much more favorable safety profile than their non-SIN counterparts.163 Recently, Montini and colleagues dissected the contribution of vector design and viral integration site selection to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced HSC in tumor-prone Cdkn2a−/− mouse models.164 By swapping genetic elements between γ-RVs and LVs, they have demonstrated that transcriptionally active LTRs with strong enhancer/promoter elements are major determinants of genotoxicity even in the context of an LV.164 Conversely, SIN LTRs improve the safety of γ-RVs because both SIN LVs and SIN γ-RVs are neutral in the mouse model. The safest design for LV or γ-RV to alleviate the risk of insertional mutagenesis therefore should combine a SIN LTR and a moderately active internal promoter. By comparing the genotoxicity of the LVs and γ-RVs with matched active LTRs, tenfold greater LV integration loads are required to obtain the same oncogenic risk as with γ-RVs. Therefore, the genotoxic risk of LVs is significantly lower than that of γ-RVs, when corrected for the same copy number. This is likely due to the preferential integration-site bias into cancer genes that is intrinsic to γ-RVs as opposed to LVs, even when genetic selection of cells harboring integration at oncogenes is prevented by the lack of strong transcriptional enhancers in the vector. Moreover, even when the SIN LV contained a strong internal promoter/enhancer, no genotoxic effects were apparent, at least in this model. This is in contrast with the results of other models showing the ability of SIN γ-RVs with strong internal enhancer/promoters to transform cells in vitro and in vivo.164,165 Though this may possibly reflect actual differences in insertion patterns between LVs and γ-RVs, it cannot be excluded that there may be other factors that limit the sensitivity of this tumor-prone model. Indeed, the intrinsic genomic instability in the Cdkn2a−/− mouse model creates a high background of spontaneous tumors that may not be ideal to assess the impact of weak genotoxic vectors. This justifies the need for additional and more sensitive models to assess genotoxicity. Indeed, using a sensitive cell culture assay and a series of SIN vectors, it was recently demonstrated that LV insertion pattern was approximately threefold less likely than the γ-RVs to trigger transformation of primary hematopoietic cells.165 However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents common insertion sites for both LVs and γ-RVs, despite major differences in their integration mechanisms. Most importantly, SIN LV containing internal tissue- or lineage-specific promoter/enhancers are likely to be safer for gene therapy applications by reducing the risk of insertional oncogenesis. For instance, LV SIN vectors containing globin regulatory elements have an improved safety profile consistent with the lack of genotoxic effects in in vitro immortalization assays.138 Moreover, the β-globin LV currently in clinical trials, which also includes a pair of 250-bp cHS4 insulator elements in the LTRs, did not accelerate tumorigenesis in a tumor-prone mouse model after transplantation of transduced HSC.76 Furthermore, the globin regulatory elements failed to activate the LMO2 proto-oncogene, at least in lymphoid cells, following a specific targeting strategy to direct integration of the globin construct into the LMO2 locus.166 However, distant gene activation by integrated globin vectors in clonal erythroid spleen colonies has been reported, suggesting that there is still a finite risk of insertional oncogenesis, even when internal tissue- or lineage- specific regulatory elements are used.167 Consequently, the residual oncogenic risk of tissue-specific LVs would likely manifest itself primarily in those tissues or lineages in which the tissue- or lineage-specific regulatory elements are active and to a lesser extent in ectopic cell types.

Therefore, LV integration profiles and/or oncogenic risks may depend on several confounding variables including the vector copy number, the target cell type, the proliferation and/or activation status of the target cells, the nature of the transgene itself, the vector design (SIN versus non-SIN, choice of promoter/enhancers), underlying disease and possible selective advantage of rapidly growing cells, protocol-specific cofactors, and finally the intrinsic genotypic variation of the model animals and the treated patients. This implies that under “permissive” conditions, it may be possible to uncover insertional oncogenic events that can be ascribed to the LV, as suggested by the discovery of common insertion sites using sensitive in vitro genotoxicity assays, described above.165 It is therefore prudent to adopt a conservative approach and optimize the experimental parameters to the extent that it results in improved efficacy while at the same token the risk of insertional oncogenesis is reduced as much as possible.

Conclusions and Perspectives

The conversion of the highly pathogenic HIV-1 into an efficient and relatively safe gene delivery vector serves as a testimony to this impressive journey that has been undertaken. LVs have now become commonplace in experimental research. Proof of concept has been established in preclinical animal models that LVs could treat or cure disease. LVs have now moved beyond the preclinical stage into the clinical arena with multiple gene therapy trials ongoing or approved. There are several clinical trials ongoing or being prepared based on the use of LVs for the treatment of β-thalassemia, ALD, Parkinson's disease, Wiskott–Aldrich syndrome, and AIDS.76,83,167,168,169,170,171,172,173 The trial conducted by Levine et al. evaluated a conditionally replicating LV expressing an antisense mRNA against the HIV envelope.174 Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single intravenous infusion of gene-modified autologous CD4+ T cells was well tolerated in all patients and immune function improved in four subjects. LV-mediated gene therapy of HSCs can provide clinical benefits in ALD, a severe demyelinating disease due to a defect in ABCD1. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with an LV encoding wild-type ABCD1, and then reinfused into the patients after they had received myeloablative treatment. Over a span of 24–30 months of follow-up, polyclonal reconstitution was detected with 9–14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that HSCs were transduced in the patients. Beginning 14–16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT.59 Despite the progress made in LV technology and the demonstration in multiple preclinical studies that animal models suffering from the cognate human disease can effectively be treated by LV-based gene therapy, there are still significant challenges ahead. In particular, the performance of LVs in patients may not necessarily mimic what has been observed in mouse models. Therefore, there is a need to conduct preclinical studies in large animal models to help bridge the gap between early proof-of-concept preclinical studies and clinical trials. It will be critically important to assess the adaptive and innate immune responses following LV-based gene therapy in subjects enrolled in clinical trials, particularly because the immune system poses a significant challenge for in vivo gene therapy with other viral vector systems. However, the lack of pre-existing immunity to vector components in most subjects may give LVs a possible advantage over other vector systems derived from viruses that are more widespread in the human population, such as AAV or adenovirus. Moreover, though the safety profile of SIN LVs has significantly improved over that of γ-RVs, integration can still potentially result in oncogene activation and/or inactivation of tumor suppressor genes. Follow-up studies will therefore be needed to assess the long-term safety and stability of LV-mediated gene therapy in patients. Nevertheless, our knowledge of the consequences of LV-mediated gene transfer is expanding rapidly and the technology is evolving providing an attractive alternative to other vector systems. Though it would seem unrealistic to expect that LV will serve as the ultimate “magic bullet” for gene therapy, the LV technology platform provides robust ammunition in the quest to develop improved therapeutics to treat otherwise incurable disease.


Part of this work presented in this review was supported by FWO, IWT, EU FP7 (PERSIST) and VIB. J.M. is supported by a grant from EU FP7 (PERSIST). We thank Christopher Baum for his useful comments.


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