Lentiviral vectors for cancer immunotherapy: transforming ... - Nature

Gene Therapy (2007) 14, 847–862 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt

REVIEW

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics K Breckpot1, JL Aerts1 and K Thielemans Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium

Lentiviral vectors have emerged as promising tools for both gene therapy and immunotherapy purposes. They exhibit several advantages over other viral systems in that they are less immunogenic and are capable of transducing a wide range of different cell types, including dendritic cells (DC). DC transduced ex vivo with a whole range of different (tumor) antigens were capable of inducing strong antigen-specific T-cell responses, both in vitro and in vivo. Recently, the administration of lentiviral vectors in vivo has gained substantial interest as an alternative method for antigenspecific immunization. This method offers a number of advantages over DC vaccines as the same lentivirus can in principle be used for all patients resulting in a significantly

reduced cost and requirement for considerably less expertise for the generation and administration of lentiviral vaccines. By selectively targeting lentiviral vectors to, or restricting transgene expression in certain cell types, selectivity, safety and efficacy can be further improved. This review will focus on the use of direct administration of lentiviral vectors encoding tumor-associated antigens (TAA) for the induction of tumor-specific immune responses in vivo, with a special focus on problems related to the generation of large amounts of highly purified virus and specific targeting of antigenpresenting cells (APC). Gene Therapy (2007) 14, 847–862. doi:10.1038/sj.gt.3302947; published online 22 March 2007

Introduction

melanoma tumors. Each of these antigens can give rise to a number of epitopes that will be presented by the tumor in major histocompatibility complex (MHC) class I or class II molecules and can be recognized by cytotoxic CD8+ T cells or helper CD4+ T cells, respectively, that were productively primed in the secondary lymphoid organs.4 Extensive research into these TAA and immune responses directed against them has instigated attempts for the manipulation of the antitumor immune response by actively trying to stimulate immunity against these TAA and consequently against the tumor itself. Thus, encouraged by the immense success of prophylactic vaccines, during the last two decades several approaches of therapeutic vaccinations have been developed as an alternative treatment for cancer. As dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) of the immune system, one of the most well-studied vaccination approaches is the use of ex vivo-generated DC loaded with TAA in one form or another to boost/induce antitumor immune responses in cancer patients. Several approaches have been studied for loading DC with antigen, the description of which is outside the scope of this review. Broadly speaking, these can be divided in viral and nonviral methods. Although cancer cells can potentially be distinguished from normal cells through a whole arsenal of antigens with highly restricted expression, these are still considered as self-antigens by the immune system. Thus, several tolerogenic mechanisms as well as different modes of active inhibition will impede a productive

The concept of a sentinel role for the immune system is longstanding and was formulated by Ehrlich in his ‘immune surveillance hypothesis’.1 Instrumental to this idea is the capacity of the immune system to distinguish ‘self’ from ‘nonself’ and to eliminate the latter without damaging the former. Although this concept was originally devised for explaining immune responses against foreign invaders, it was later refined by Burnet and Thomas,2 who applied it to antitumor immune responses in their so called ‘tumor surveillance hypothesis’. Herein they propose a model where tumor cells are recognized as foreign by the immune system and subsequently specifically eliminated without damaging their healthy counterparts, much in the same way as for virus infected cells, thus avoiding autoimmunity. The identification of antigens that are specifically expressed by the tumor but not by normal tissues offered a rationale for this hypothesis.3 These so-called tumorassociated antigens (TAA) can be divided in several groups according to their expression pattern. As melanoma constitutes the most immunogenic cancer, most of the TAA known to date were identified from Correspondence: Dr K Breckpot or Dr J Aerts, Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel, Laarbeeklaan 103/E, Brussels 1090, Belgium. E-mails: [email protected] or [email protected] 1 These authors contributed equally to this work. Received 11 October 2006; revised 1 February 2007; accepted 2 February 2007; published online 22 March 2007

Lentiviral vectors in cancer immunotherapy K Breckpot et al

848

antitumor immune response. A manifestation of this phenomenon is the presence of high numbers of cytotoxic T lymphocytes (CTL) in the tumor and the periphery without measurable tumor regression.5 Taken together, for any therapeutic vaccination strategy to be successful, it is crucial not only to induce a specific immune response against the tumor but also to overcome intrinsic tolerogenic and suppressive mechanisms. Recently several mechanisms have been described that contribute to this lack of functional antitumor reactivity. One of the paradigms of immunology is that a productive T-cell immune response requires specific recognition of an MHC-peptide complex by the T-cell receptor (signal 1) along with adequate signaling through costimulatory molecules (signal 2). Recently, more and more data indicate that to establish strong antitumor responses, an inflammatory environment (signal 3) is required alongside functional recognition of T cells by APC. This can be achieved by strong activation of the innate arm of the immune system, in particular through toll-like receptors (TLR). Yang et al.6 recently showed that in order to break tolerance of tumor-specific CTL, persistent TLR ligation should be provided alongside antigen-loaded mature DC. Lang et al.7 showed that even in a transgenic mouse model where the antigen is highly expressed in the target organ (in this case the pancreas) adoptively transferred CTL were not able to lyze actually the target cells. Only concomitant TLR ligand administration induced frank diabetes. Besides, a lack of sufficient stimulation to break tolerance, several inhibitory mechanisms, have been identified that are responsible for active quenching of the antitumor immune response. Many of these mechanisms are based on a paracrine secretion of cytokines either by the tumor or by other cells, including members of the interleukin (IL)-10 family and transforming growth factor-b. However, a new subtype of CD4+ T cells has recently emerged, which shows a contactdependent inhibitory capacity toward effector T cells.8 In mouse studies, we and others have shown that depletion of CD4+CD25+ regulatory T cells (Treg) greatly enhances the antitumor response.9,10 Furthermore, in tumor biopsies from ovarian cancer patients, significant numbers of Treg were found which were associated with a high death hazard and reduced survival.11 Therefore, specific removal of Treg inhibition is important to boost the antitumor response of cytotoxic CD8+ T cells. It has recently emerged that signaling through certain combinations of TLR on DC not only provides a synergy with respect to the production of cytokines such as IL-12,12,13 but also offers protection from Treg inhibition.14 In light of these data, it is becoming evident that it will not be enough to merely generate mature DC efficiently presenting TAA-derived epitopes, but that additional signals need to be provided to overcome inherent mechanisms of tolerance. Although lentiviral vectors are devoid of virulence factors, they might still be a source for activation signals for the innate immune system. Together with providing sufficient amounts of antigen for T-cell priming to DC, lentiviral vectors might also contribute certain inflammatory signals (e.g., through their viral RNA). Thus, the capacity of lentiviral vectors to induce long-term transgene expression as their chief advantage might be eclipsed by their ability to offer significant amounts of ‘signal 3’.

Gene Therapy

In this review, we will first describe lentiviral vectors for the transduction of DC in vitro. Ultimately, we will describe the potential use of lentiviral vectors as a vehicle for direct therapeutic vaccination.

Lentivirus-based vectors The development of retrovirus- and lentivirus-based vectors The use of gene delivery vectors based on retroviruses was introduced in the early 1980s by Mann et al.15 The family of the retroviridae consists of single-stranded RNA (ssRNA) viruses, which are spherical, measure about 80–120 nm in diameter, have a mass of B2.5 105 kDa and represent a density of 1.16 g/ml in sucrose density gradients.16 These ssRNA viruses replicate through a double-stranded DNA intermediate, integrating their cargo into the host genome where they express viral RNA during the lifetime of the cell. The retroviridae are generally divided in two categories – simple and complex – based on their genome organization. The most commonly used oncogenic retroviral vectors are based on Moloney Murine Leukaemia Virus (MLV) and have a simple genome. From this genome, the polyproteins, gag, pol and env are required in trans for viral replication and packaging. Required in cis are the 50 and 30 long-terminal repeat (LTR), the integration sequences as well as the packaging site c, the transport RNA-binding site and finally additional sequences involved in reverse transcription. To generate replication-deficient retroviral vectors, the gag, pol and env genes are replaced with an expression cassette. The major advantage of retroviral vectors in view of inducing antitumor responses is their lack of immunogenicity due to the removal of most genes encoding viral proteins, thus enabling repeated use of these vectors for immunization. However, there are some limitations associated with oncogenic retroviral vectors. The most important are: (1) the instability of the viral particle,17 (2) the low viral titers18 and (3) the inability to transduce nondividing cells.19 To overcome these shortcomings, lentivirus-based vectors were developed.20 Lentiviruses constitute a subclass of retroviruses of which mainly simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) have been extensively studied. They display the same basic genomic organization as oncogenic retroviruses,21 but are more complex due to the presence of accessory genes – vif, vpr, vpu, nef, tat and rev – some of which play crucial roles in virus replication (Figure 1).22 Unlike retroviruses, lentiviruses are capable of transducing quiescent cells.23 For HIV, this process appears to be facilitated by the following viral proteins: (1) the integrase protein,24,25 (2) the matrix protein from gag,26 (3) the accessory protein vpr27 and (4) the central polypurine tract (cPPT).28 As a result of their capacity to transduce nondividing cells – although for some cell types, progression through the cell cycle from G0 to G1b is necessary for efficient, complete reverse transcription of the viral genome29 – lentiviral vector development has received much interest. In analogy with the design of oncogenic retroviral vectors, the design of lentiviral vectors is based on the separation of cis- and trans-acting sequences. Generally,


lentiviral particles are generated through transient transfection of three plasmids in the human embryonal kidney (HEK) 293T producer cell line.30 The plasmids in question are: (1) a packaging plasmid, (2) a transfer plasmid and (3) an envelope-encoding plasmid. The lentiviral particles are divided into ‘generations’ according to which packaging plasmid was used for virus production. The first-generation packaging construct provided the entire gag and pol sequences in trans to enable packaging of the transfer construct and contains the viral regulatory genes tat and rev along with the accessory genes vif, vpr, vpu and nef. Identification of the

gag

5' LTR

HIV genes that are necessary for virulence, but disposable for transfer of the genetic cargo, allowed the engineering of multiple-attenuated packaging systems.22,31 In these second-generation packaging systems, up to four accessory genes (vif, vpr, vpu and nef) were removed without negative effects on yield or infection efficiency in most cell types. These minimal packaging systems gained in safety, as any replication-competent lentivirus (RCL) that was still generated would be devoid of all virulence factors. Safety was further improved by engineering split-genome packaging systems (third generation), where the rev gene is expressed from a separate plasmid. Finally, tat could be removed by placing a strong constitutional promoter in the 50 LTR of the transfer vector.32 The transfer plasmid consists of an expression cassette as well as the HIV cis-acting factors necessary for packaging, reverse transcription and integration. Engineering of self-inactivating (SIN) transfer vectors by deleting the U3 region of the 30 LTR33,34 not only further minimized the risk of emergence of RCL, but also decreased problems related to promoter interference.35 Nuclear import of the transfer construct was improved by including the cPPT and its central termination sequence, together forming a triple helix (TRIP). This structure mediates the transport of the preintegration complex through the nuclear pores. TRIP/cPPT-containing transfer vectors yield higher virus titers and provide enhanced transgene expression in the target cells.28,36 Addition of post-transcriptionally active elements, such as the woodchuck hepatitis B post-transcriptional regulatory element (WPRE), represents another strategy to improve the lentiviral vector design. WPRE may improve gene expression by modification of polyadenylation, RNA export or translation (Figure 2).37 The third plasmid required for lentivirus production, provides a heterologous envelope, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), which allows the formation of mixed particles or pseudotypes, displaying the tropism of the virus from which the envelope is derived.38 This topic is discussed in more detail in the following section.

3' LTR

pol ψ

env MLV vif

gag

5' LTR

3' LTR

pol

ref tat

ψ

vpr

vpu

nef

env

HIV-1 promoter

polyA

∆ψ accessory genes

gag

RRE

pol regulatory genes 1st generation packaging plasmid gag

RRE pol regulatory genes 2nd generation packaging plasmid

gag

RRE pol

849

Rev 3rd generation packaging plasmid

Figure 1 Schematic representation of the native MLV and HIV-1 genome, as well as the different lentiviral packaging generations. Adapted from Delenda et al.166

Altering the tropism of lentiviral vectors by pseudotyping and transcriptional regulation Pseudotyping of lentiviral vectors. Among the first and still most widely used glycoproteins for pseudotyping

constitutive inducible tissue-specific

other promotor Tat-independent transcription U3 promotor Tat-dependent transcription

cPPT/CTS R

U5

ψ

WPRE

Fsgag promoter transgene

U3 non-SIN vector

R

U5

∆U3 SIN vector

IRES bi-cistronic expression vector Figure 2 Optimization of the lentiviral transfer vector. Adapted from Delenda et al.166 Gene Therapy


850

lentiviral vectors is VSV.G. Lentiviral vectors pseudotyped with VSV.G offer significant advantages in that VSV.G appears to interact with a ubiquitous cellular receptor on cells, endowing the vector with a broad host-cell range.39,40 Furthermore, VSV.G confers high vector particle stability, allowing downstream processing of the viral particles.41 Possible shortcomings associated with the use of VSV.G are (1) its toxicity when constitutively expressed42 and (2) its inactivation by serum complement.43 However, the latter can be overcome by using chemically modified VSV.G pseudotypes, for example, polyethylene glycol modified particles.43,44 There is an ever growing list of alternative glycoproteins for pseudotyping lentiviral vectors, each with its advantages and disadvantages. This list includes the glycoproteins from rhabdoviridae, arenaviridae, flaviviridae, paramyxoviridae, baculoviridae, filoviridae and retroviridae. These glycoproteins have been successfully used for pseudotyping HIV-1 and/or SIV-derived vectors. Importantly, all of these glycoproteins preferentially interact with certain cell types, and can thus be applied to obtain cell-specific transgene expression. An overview is given in Table 1.

Transcriptional regulation and targeting: the promotor. For many applications, the potential to regulate or obtain cell-specific expression is appealing. Therefore, several groups have focused their research on the use of inducible or tissue-specific promoters. Among the most widely used induction systems, the tetracycline-based approach has been adapted for transcriptional regulation of transfer vectors.45–47 For

Table 1

gene-therapy applications, an inducible vector dependent on the delivery of the antibiotic (tet-on) is preferred over one based on gene silencing, which would necessitate constant antibiotics treatment in patients unless transgene expression is required, whereas in the tet-on system, antibiotics are only given when the gene needs to be turned on. With the exception of two reports,46,48 all the tetracycline-inducible HIV-1 vectors listed in the literature were constructed using the tet-on system.49,50 The authors showed that the tetracycline approach applied to HIV-1 gene transfer vectors allows inducible expression, that is dose-dependent and rapidly switched on and off. To direct specifically transgene expression in particular cell types or organs, several groups have used tissuespecific promoters. To date, specific gene expression has been described for several cell types. Specific targeting of cells from the erythroid lineage51 was obtained with the ankyrin-1 promoter. Endothelial cells52 have been successfully targeted using the angiopoietin receptor Tie2. Cells from the central nervous system53 showed a higher transgene expression on transduction with enolase promoter containing lentiviral vectors when compared to the cytomegolovirus (CMV) containing viruses both in vitro and in vivo. Use of the rhodopsin promoter in an HIV-1-based vector has not only led to photoreceptorspecific expression, but also to higher expression levels than with constitutive promoters.54 Liver-specific expression has been achieved using the albumin promoter.55 Moreover, Oertel et al.56 observed that long-term expression in rat liver was achieved when the transgene was driven by the liver-specific albumin promoter but was silenced when the CMV promoter was used.

Overview of the envelopes used for pseudotyping of lentiviral vectors

Family

Genus

Vector

Target

Reference

Rhabdoviridae

Vesiculovirus VSV Lyssavirus Rabies virus Mokola virus

HIV-1, SIV HIV-1

Broad host range tropism Neuronal cells

30,167,168

Arenaviridae

Arenavirus Lymphocytic Choriomeningitis virus

HIV-1, SIV

Cancer cells and astrocytes

171,172

Flaviviridae

Hepacivirus Hepatitic C virus

HIV-1

CD81 positive cells, such as hepatoma cells

173,174

Paramixoviridae

Respirovirus Sendai virus

HIV-1 SIV

Hepatocytes

175

Baculoviridae

Nucleopolyhedrovirus Fowl plague virus

HIV-1 SIV

Broad host range tropism, poor transduction of hematopoietic cells

176,177

Filoviridae

Filovirus Ebola virus Marburg virus Lassa virus

HIV-1

Lung cells and myocytes

74

Retroviridae

Alpharetrovirus Jaagsiekte sheep, Ross River, Semiliki Forest, Sindbis Virus Gammaretrovirus MLV Gibbon ape Leukaemia virus

HIV-1

Lung cells

178,179

HIV-1 SIV

Cancer cells

168,172,180

Gene Therapy

169,170


In view of anticancer therapy, it is important to elaborate on the use of specific promoters to restrict transgene expression to cancer cells and cells involved in the antitumor immune response. Human hepatocarcinoma cells have been transduced with lentiviruses containing a suicide gene under the control of the a-fetoprotein promoter resulting in specific destruction of these malignant cells.57 Furthermore, a patient-derived prostate-specific antigen (PSA) promoter inserted into a lentiviral vector has driven efficient transgenic activity in prostate cells with satisfactory efficacy and specificity both in vitro and in vivo.58 More recently, a PSA promoterbased HIV-1 vector has been used to deliver the diphtheria toxin A gene into prostate cancer cells, resulting in tissue-specific eradication of prostate cancer cells in cell culture and in a mouse tumor model.59 As discussed in the general introduction, APC and in particular the DC constitute an interesting cell population in view of immunotherapy. To achieve APC-specific transgene expression, Cui et al.60 took advantage of the fact that MHC class II genes are expressed selectively in APC and highly in DC after differentiation and maturation. Using the non obese diabetic (NOD)/severe combined immunodeficient (SCID) mouse engraftment model, they demonstrated selective expression of the transgene in MHC class II positive human cells with an HIV-1 SIN vector harboring the human MHC class II-specific HLA-DRa promoter.

Downstream processing of lentiviral vectors Production of lentiviral vectors. For small-scale experimental purposes, lentiviral vectors have been often generated through transient transfection of the producer cell line HEK 293T, with plasmids encoding for the structural proteins, enzymes, envelope and viral RNA to be packaged. However, upscaling the production in a reproducible and standardized fashion will be essential for lentiviral vectors to enter the clinic. Important parameters influencing large-scale vector production are the choice of producer cell line, media and type of vessel for cell culture.61 The development of producer cell lines will be discussed in the following section. Concerning the cell culture vessel, advantages of reactors, such as stirred tanks, over multiple systems, such as T-flasks, cell factories and roller bottles include (1) scalability, (2) use of perfusion methods and (3) access to control units, allowing close monitoring of the culture conditions. Until now, it has been described that the best results are obtained in reactors that allow high cell density.62,63 Comparison of stationary versus different microcarrier cultures and the latter versus roller bottle cultures have been performed, showing that in both comparisons, the microcarrier cultures were superior for the production of high-titer vector stocks, generating titers of about 107 TU/ml, which are approximately a factor 2 higher than the traditional producer systems. Producer cell lines for lentivirus production. Although numerous groups reported attempts to generate stable, well-characterized packaging cell lines,64–69 none of these systems are currently widely used, probably because the overall benefit compared to transient transfection remains relatively modest. The major problem with the development of such cell lines is the cytotoxicity of

constitutive expression of high amounts of HIV-1 proteins, such as pol, gag, vpr, tat, rev and protease.70–73 Furthermore, the constitutive expression of the widely used envelope protein VSV.G is detrimental to mammalian cells.41 Engineering inducible expression under the control of a drug-dependent element of both the viral proteins and VSV.G has resulted in stable packaging cell lines, resulting in acceptable vector yield, varying between 105 and 107 TU/ml.64–67 Alternatively, replacing VSV.G with glycoproteins displaying an equally broad tropism but that do not lead to producer cell death, such as baculovirus envelope,74 gammavirus envelope68 or alphavirus envelope,69 has enabled the establishment of stable packaging cell lines. However, it is obvious that only virus production with stable packaging cell lines can meet the demands for clinical use when it comes to reproducibility and standardization.

851

Concentration and purification of lentiviral particles. Large-scale, high-titer vector production is challenging due to the lack of simple procedures for rapidly processing large volumes of cell culture supernatant. Traditionally, physical concentration methods, including ultracentrifugation and ultrafiltration of virus-containing cell culture supernatants, have been used for this purpose.41,75,76 However, these approaches are limited in terms of their capacity to handle large volumes, thus making this procedure extremely tedious. Low-speed overnight centrifugation has been used to process volumes in excess of one liter.77 However, this low-speed approach is time consuming. Thus, there is an emerging need for quick, reproducible and less laborious procedures that rapidly reduce the volume of the cell culture supernatant to be processed. Pham et al.78 and Zhang et al.79 have both described a precipitation method to concentrate lentiviral vectors involving the coprecipitation of viral supernatants with calcium phosphate and poly-L-lysine, respectively. In this way, volumes were markedly decreased, but recovery of infectious virus was rather low (32–50%). One problem the methods outlined above have in common is that cellderived components are concentrated along with the vector particles leading to potential immune and even inflammatory responses.80 Thus, these approaches generate rather impure virus preparations and additional steps, including chromatography-based approaches are needed to remove contaminating host-cell components from the virus preparation. Recently, methods based on anion exchange chromatography of HIV-1 vectors pseudotyped with VSV.G and baculovirus glycoprotein 64 have been successfully established.81–83 Titer determination. To standardize the number of viral particles administered to patients, it is crucial to accurately titrate the virus for each individual vector production. However, titering procedures of lentiviral vectors are still plagued by large intra- and inter-assay variation. Furthermore, a whole range of different methods is used by individual laboratories. Some of these methods are based on the number of vector particles present in a virus stock, whereas others are derived from the number of provirus copies in transduced target cells. Virus particle numbers can be determined using real-time polymerase chain reaction (PCR) based on strong-stop cDNA present in virions84 or Gene Therapy


852

using DNA extracted from transduced cells.85,86 Alternatively, the amount of virus proteins present in virus cores such as p24 gag are determined by enzyme-linked immunosorbent assay to arrive at relative particle titers.87 Titration assays based on vector-encoded reporter gene expression using fluorescence-activated cell sorting analysis of cells transduced with varying amounts of the virus have been widely used.88 An important factor is the cell line used for titration, as receptors for a given glycoprotein may vary from cell line to cell line. Frequently, a cell line (such as HeLa or 293 cells) is chosen because it is considered generally permissive to infection rather than representing the natural tropism of the virus from which the glycoprotein was derived. Taken together, as each method produces different results, one should pay attention to the quantification method used before comparing results.

Lentiviral transduction of DCs Efficient transduction of human DC with transgenic vectors has been challenging for several reasons. Human DCs are usually generated from peripheral blood-derived quiescent CD14+ progenitors or from mitotically hypoactive primitive CD34+-derived progenitors cells (hematopoietic stem cells). Therefore, the capacity of lentiviral vectors to transduce quiescent and nondividing cells turned out to be an important asset for DC transduction. The first successful transduction of human monocytederived DC (MoDC) with lentiviral vectors was described by Unutmaz et al.89 in 1999. Since then, several research groups have reported successful transduction of monocyte-derived,88,90–93 CD34+-derived human DC60,94–96 and mouse bone marrow-derived (BM) DC88,97–99 with varying efficiencies. Transgene expression was found to be stable in human MoDC31,88,91,96 and CD34+ DC.94 However, for murine BMDC, the kinetics are somewhat more complicated. When tNGFR is used as a reporter gene, around 83% of cells are tNGFR positive on day 2 after transduction. Six days after transduction, this percentage decreases to on average 30%. However, with eGFP as a reporter gene, the opposite is observed. The expression of eGFP gradually increases from around 47% on day 2 to 65% on day 6. Using eGFP-encoding lentiviral particles produced in cells that were simultaneously transfected with a nonviral eukaryotic expression vector carrying tNGFR and vice versa, our group demonstrated that nonviral tNGFR is delivered by eGFP-encoding lentiviruses on day 1 after transduction (37% tNGFR positive). The tNGFR positivity decreases with time and almost disappears by day 3 after transduction. This process has been described as pseudotransduction.100 No pseudotransduction with nonviral eGFP delivered by tNGFRencoding lentiviruses was observed.101 Importantly, the variability in transduction efficiency among different reports reflects the diversity in DC sources, techniques and vectors used for transduction. Besides efficient expression of a transgenic protein in lentivirally transduced DC, it is of paramount importance that transgene-derived peptides are efficiently processed and presented on the DC surface to prime efficiently and activate antigen-specific T cells. The first experiments to test the antigen-presenting capacity of lentivirally transduced DC were performed with established T-cell lines. Gene Therapy

Both human and murine lentivirally transduced DC were able to activate established CD4+ and CD8+ T-cell lines/ clones specific for epitopes derived from various relevant TAA, such as MAGE-3,102 Melan-A/MART-192,96 and tyrosinase93 in the human system and for the surrogate TAA ovalbumin (OVA)88,103,104 as well as for TRP-297 in the murine system. These data show that DCs are capable of efficiently processing and presenting the lentivirally delivered transgene resulting in activation of established T cells. More importantly, several groups reported in vitro priming of naive T cells against TAA using lentivirally transduced human DC. Thus, Firat et al.92 showed priming of CD8+ T cells in bulk following in vitro stimulation with melanoma poly-epitope-transduced MoDC. They observed amplification of tetramer-positive CD8+ T cells, which could specifically lyze gp100 and MART-1 peptide-pulsed targets. Our group showed priming of both CD8+ and CD4+ T cells against the poorly immunogenic melanoma antigen MAGE-A3 after in vitro stimulations with DC transduced with a lentivirus encoding the fusion protein Ii80/MAGE-A3.102 Subcloning of one of the CD8+ T-cell clones enabled the identification of a novel HLA-Cw7-restricted peptide. A number of groups evaluated the use of lentivirally transduced DC in mouse models as an immunotherapeutic agent against cancer in vivo. The immune response that was induced was characterized and tested for protection against tumor growth. Our group showed that immunization with BMDC lentivirally transduced with OVA induced a strong CTL response, resulting in specific lysis of OVA-expressing tumor cells after in vitro restimulation. Mice immunized with OVA-transduced DC were also capable of specifically eliminating autologous OVA peptide-pulsed spleen cells in vivo.104 Importantly, the resulting CTL response was protective against a subsequent challenge with a lethal dose of OVA-expressing B16 melanoma cells and slowed down the outgrowth of pre-existing tumors.103,104 As these studies used exogenous and strong immunogenic antigens, they may overestimate the effects of lentivirally transduced DC against endogenous TAA. Nevertheless, Metharom et al.97 also observed strong protective immunity against subsequent challenge with B16 melanoma after immunization with BMDC transduced with the endogenously expressed TAA, TRP-2. Immunization with TRP-2-transduced BMDC also improved survival of mice with pre-existing tumors. Taken together, these studies show that lentivirally transduced DC can present tumor antigen-derived peptides, and that in mouse models, the immune response mounted by lentivirally transduced DC is protective against subsequent tumor challenge and induces regression of pre-established tumors. This suggests that lentivirally transduced DC might be effective in therapeutic treatment of melanoma and other tumors.

Direct administration of lentiviral vectors Immune responses and inhibition of tumor growth after direct administration of tumor antigen-encoding lentiviral vectors On the basis of the successes of virally mediated genetherapy approaches, where viral vectors are used to


deliver a corrected version of a gene to the appropriate cells, the use of TAA-encoding lentiviral vectors has been explored for generating antitumor responses in vivo. The premise of this approach is that lentiviral vectors when administered directly in vivo are able to specifically transduce APC and in particular DC. These DC will then mature, present antigen and migrate to the lymph node where they can effectively prime T cells that will in turn migrate to the tumor site. Several groups have presented evidence for this selective in vivo transduction of DC. VandenDriessche et al.77 were the first to show that APC within the spleen cells are selectively transduced on in vivo administration of lentiviral vectors. Palmowski et al.105 also showed that a small percentage of GFP positive CD11c+ cells (0.3 to 0.4%) were recovered from the spleen after in vivo administration of lentivirus. When we tried to analyze the cells that were transduced within the draining lymph node on lentiviral administration we only found a very small percentage (o0.1%) of GFPpositive cells.104 When dissecting the cell populations that were transduced by analyzing frozen draining lymph node sections by immunofluorescence, Esslinger et al.98 found that the majority of the transduced cells were CD11c+ DC. Nevertheless, about 10% of the cells that were transduced could not be identified with the markers used in this analysis. Recently, He et al.106 demonstrated that DC residing in the skin and not lymph node resident DC are predominantly transduced upon footpad injection of eGFP-encoding lentivirus. Furthermore, they showed that the lentivirally infected skin DC are responsible for priming of T cells in the lymph nodes. The ultimate measure for the value of direct administration of lentivirus in tumor immunology is the degree of antigen-specific CTL induction. Using HLA-Cw3 as a model antigen, antigen-specific CTL responses could be generated on direct administration of lentiviral vectors.98 When comparing immune responses generated by direct administration of lentivirus with vaccination with DC that were transduced with lentivirus ex vivo, superior immune responses were generated by direct administration of lentivirus both in terms of strength and longevity. Similar results were obtained in HLA-A*0201 transgenic mice using a lentivirus encoding a minigene containing the dominant MART-1/Melan-A HLA-A*0201 epitope. Using OVA as a model antigen, we confirmed that direct administration of lentiviral vectors is superior to vaccination with ex vivo-transduced DC, both in terms of the number of interferon (IFN)-g producing CTL as determined by ELISPOT in vitro and the lytic capacity of CTL determined by an in vivo CTL assay. Moreover, memory CTL responses were significantly stronger with direct lentivirus administration. Other studies have also shown potent immune responses on direct in vivo administration of lentiviral vectors encoding relevant tumor antigens such as NY-ESO105 or TRP-2,107 although no direct comparison with ex vivo-transduced DC was performed here. Recently, Chapatte et al.108 compared direct administration of a full length human MART-1/Melan-Aencoding lentiviral vector with MART-1 peptide–adjuvant immunization in a HLA-A*0201 transgenic mouse model and found that the anti-MART-1 immune response was higher for the lentivirus injection than for the peptide-adjuvant vaccination.108 Although the generation of a specific CTL response is a convenient read-out for the success of a vaccination

strategy, there are many examples of discrepancies between immune responses and antitumor responses.109 Therefore it is crucial not only to evaluate the TAAspecific immune response but also the influence of the treatment on tumor growth. Whereas Rowe et al.110 showed significantly improved protection of direct administration of an OVA-encoding lentiviral vector against a subsequent tumor challenge, no data was shown about the therapeutic efficacy of this treatment. Using a similar OVA-encoding lentiviral vector we could show that direct administration of lentiviral vectors offers increased protection to a subsequent tumor challenge compared to DC vaccination and a significantly improved survival of tumor bearing mice. This increased survival was only marginally dependent on the amount of transducing units that were administered to the animals as 2 106 and 107 TU resulted in similar survival curves. In contrast, increased numbers of DC (5 105 vs 105) yielded a significantly better survival.104 In another study using TRP-2 as a tumor antigen, improved survival of mice receiving lentivirus-encoding TRP-2 was reported compared to irrelevant lentivirus.107 Taken together, there is substantial evidence for the induction of specific CTL responses by TAA-encoding lentiviral vector administration in vivo. There are also important indications that this treatment can induce protective immunity and our work also shows that direct lentivirus administration results in increased survival, not only compared to controls but also compared to vaccination with ex vivo-transduced DC.

853

Immunogenicity of lentiviral vectors Although the currently used lentiviral vectors are – when compared to other viral systems – almost devoid of viral proteins, as described in the previous section, administration of lentiviral vectors does not provoke immunological tolerance, but instead elicits powerful CTL responses against transgene-encoded proteins. This suggests a certain degree of immunogenicity of lentiviral vectors or components present in virus preparations, leading to activation of innate viral-sensing pathways and ultimately resulting in a strong adaptive immune response. It was already shown in 1997 that wild-type HIV-1, from which most recombinant lentiviruses are derived, induces cell- and antibody-mediated responses in humans.111–113 Moreover, recently it has been demonstrated in vitro that wild-type HIV-1 activates a subset of DC, the plasmacytoid DC (pDC), through engagement of TLR 7, a pattern recognition receptor (PRR) for ssRNA.114,115 On infection, these pDC produce high levels of type I IFN, which are potent antiviral cytokines that induce the maturation of pDC as well as the maturation of other DC subsets.114,116 This DC maturation, in turn, can lead to activation of the adaptive immune system. As the recombinant lentiviral vectors are HIV-derived and contain ssRNA by necessity, they may also trigger a similar innate immune response. The direct influence of lentiviral vectors on MoDC, which is the DC subset that has been most extensively used for both in vitro experiments and DC vaccination of cancer patients, has been previously studied, but remains controversial. Gruber et al.31 reported that transduction of immature MoDC with lentiviruses at multiplicity of injection (MOI) 5 does not result in phenotypical or Gene Therapy


854

functional maturation, whereas Tan et al.117 described that transduction of immature MoDC with a MOI of 500 results in upregulation of adhesion, costimulatory and HLA molecules. Furthermore, Tan et al. showed that these DC display enhanced allo-stimulatory capacities and display an altered cytokine secretion pattern. Our data indicate that transduction of DC at low MOI (1.5) results in considerable transgene delivery (32712%, n ¼ 3), but does not lead to activation, whereas transduction at higher MOI (15–150) leads to phenotypical and functional maturation, confirming the results of Tan et al. (Breckpot et al., unpublished data). To elucidate the mechanisms mediating this lentivirus-induced activation, Tan et al. investigated the role of protein kinase R (PKR), and demonstrated that this cytosolic receptor, which interacts with dsRNA, an intermediate in the lentiviral replication, is phosporylated on transduction at high MOI. This was confirmed by our group (unpublished data). The phosphorylation of PKR leads to the phosphorylation of IkB and subsequent activation of the nuclear factor kB, and thus, the transcription of several genes with antiviral activity,118,119 such as IFN-a secreted by MoDC on lentiviral transduction,31 Breckpot et al., unpublished data). Toll-like receptors are another important PRR for sensing viral infections.120 Therefore, we evaluated the activation of TLR 2, 3 and 8 – all expressed both on blood-derived myeloid DC and in vitro-generated IL-4/ GM-CSF MoDC – by VSV.G pseudotyped, secondgeneration lentiviral vectors. In a reporter system using 293T cells transfected with each of these TLR together with a luciferase construct driven by the NF-kB promoter, we demonstrated that transduction at a MOI of 15 or higher resulted in activation of TLR 2, 3 and 8, which are probably engaged by virion components, dsRNA (a viral replication intermediate) and ssRNA (the viral genome), respectively. We further showed that this activation is TLR dependent, as lentiviral transduction of 293T cells transfected with only the luciferase construct did not result in enhanced luciferase activity. Furthermore, we demonstrated that the observed effects were an outcome of the transduction of the 293T cells, as heatinactivation of the viruses or pre-treatment of the viruses with a blocking anti-VSV.G antibody resulted in decreased luciferase activity. Although the downregulation of luciferase activity was less than for TLR 3 and TLR 8, the downregulation observed for TLR2 was unexpected, as TLR 2 – in contrast to TLR 3 and TLR 8 – is localized on the DC surface and has been documented to interact with virions.121 Assesment of the role of VSV.G on the activation of TLR 2 demonstrated that VSV.G is partially responsible for the observed TLR 2 activation. The in vitro studies described above show that MoDC are activated by VSV.G-pseudotyped lentiviral vectors. However, the activation of conventional DC in vivo has – to our knowledge – not been studied. In contrast, a role for pDC on in vivo administration of lentiviral vectors has been suggested by Brown et al.122 They show that administration of lentiviral vectors to mice triggers a rapid and transient type I IFN response. The observed effect was independent of the envelope pseudotype, but dependent on functional vector particles, suggesting the necessity of cell entry. In vitro challenge of APC suggested that pDC are responsible for this response. Although the precise mechanism of pDC activation is not

Gene Therapy

investigated, the authors suggest a role for TLR 7 based on its endosomal localization and its interaction with ssRNA. Furthermore, as reverse transcription of the viral genome occurs in the viral particle itself,123 they do not exclude the possibility that alternate DNA PRR124,125 or TLR 9 is triggered by the lentiviral vectors. They further suggest the presence of additional elements within the vector and/or target cells that are involved in triggering the innate host response, as TLR 7/9 antagonists are not sufficient to prevent the administered lentiviruses to induce a type I IFN response (unpublished data by Brown BD and Sitia G). A recent report by Pichlmair et al.126 reported on the presence of tubulovesicular structures (TVS) of cellular origin, carrying nucleic acids, including DNA plasmids originally used for lentivirus production within VSV.Gpseudotyped lentivirus preparations. These TVS act as a stimulus for innate antiviral responses, triggering TLR 9 and inducing type I IFN production by pDC. They further demonstrate that removal of these TVS markedly reduces the capacity of the lentiviral vectors to activate pDC and that these TVS alone are sufficient to stimulate pDC and act as a potent adjuvant in vivo, eliciting both T- and B-cell responses to coadministered proteins. Their results highlight the important role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations. In the Introduction, it is mentioned that more and more data indicate that the establishment of a strong antitumor response is dependent on an inflammatory environment (signal 3) alongside functional recognition of T cells by APC, and that this can be achieved by strong activation of the innate arm of the immune system, in particular through TLR. Two studies, one by Yang et al. and the other by Lang et al., clearly demonstrated that tolerance of antigen-specific CTL could be broken by persistent TLR ligation.6,7 Furthermore, it has been recently described that signaling through certain combinations of TLR on DC not only provided a synergy with respect to the production of cytokines such as IL-12, which is essential for skewing CD4+ T cells toward a Th1 phenotype,12,13 but also offered protection from inhibitory CD4+ T cells that quench the antitumor immune response.14 In addition to breaking tolerance, a productive CD4+ T-cell response is required for the induction of a powerful CTL response. Although earlier work shows that CTL responses can be induced in the absence of CD4+ T-cell help,127 several recent studies prove that CD4+ T-cell help is indispensable for generation of effective memory CTL and recall responses, even in the case of viruses that elicit potent primary CTL responses.128–130 For direct administration of lentiviral vectors, several groups have shown that both a CTL response and an antigen-specific CD4+ T-cell response can be induced.98,104,110 However, not much data is available on the role of CD4+ T-cell help in CTL induction. Esslinger et al. showed that CD4 depletion reduces primary CTL response on direct administration of lentivirus. We showed that although their was a larger requirement for CD4+ T-cell help during the primary response in case of immunization with DC transduced ex vivo compared to direct administration of lentiviral vectors, CD4+ T-cell depletion strongly reduced the capacity to mount a recall CTL response in both cases.104 Interestingly, Marzo et al.131 showed that in the case of a


VSV infection, a functional CD8+ T-cell memory response can be generated in the absence of CD4+ T cells, this in contrast to an infection with Listeria monocytogenes. These authors suggest that the difference might be due to the fact that VSV can directly infect DC whereas LM needs to be cross-presented. As the lentiviral vectors that have been used so far for direct in vivo adminisitration all have been pseudotyped with a VSV.G envelope, it needs to be further examined to what extent the CTL response is CD4+ T-cell dependent. Overall, it has become clear from the above-discussed studies on activation of MoDC and pDC by lentiviral vectors, that lentiviral vectors as they are currently produced and processed induce DC activation through TLR signaling and other mechanisms, explaining their potency as antitumor vaccine.

Toward specific targeting of DCs by in vivo lentiviral vector administration? Although recent data show that there is specificity for DC transduction in vivo when administering lentiviral vectors, this issue along with the question, which other cell types are further transduced, still needs to be explored in more detail. One of the most important questions that remain is whether it is sufficient to transduce only DC, and if yes, which subtype of DC yields better immune responses. Several approaches can be taken to analyze this more clearly. One way would be to replace the constitutively active promoters, such as CMV, CMV enhancer/chicken b-actin core promoter (CAG) and so on by DC-specific promoters. Thus, the CD11c promoter could be used to limit expression in myeloid DC,132 whereas the BDCA-2 and langerin promoter should result in exclusive expression in pDC133 and Langerhans cells,134 respectively. As the expression level of the transgene could be significantly reduced compared to constitutively active promoters, the question remains whether this will be a valuable approach. Another approach that has been attempted is to modify the viral envelope in such a way that the infectivity of the virus is restricted to or preferential for a certain cell type. Modifications can be made chemically, but recently a convenient system was described for retroviral vectors where the viral-packaging mechanism is exploited to modify the viral envelope. As the virus is generated in a producer cell line, on budding it incorporates part of the cell membrane in its envelope. If a specific receptor is overexpressed on the cell membrane of a producer cell line, then this will also be displayed on the viral envelope. When overexpressing stem cell factor (SCF) on the cell membrane of an ecotropic producer cell line, Chandrashekran et al.135,136 showed that retrovirus thus produced was able to preferentially transduce c-kit expressing human stem cells. Recently, Yang et al.137 demonstrated that this approach can also be applied for lentiviruses. This method could thus be modified to generate lentiviral vectors that can specifically target DC (or other cell types) and still yield high expression of the transgene. Receptors that could be used include CD40 ligand, which interacts with CD40, and CTLA-4, which interacts with CD80 and CD86. As many of these receptors are expressed on other cell types than DC, specificity will still be limited. Specific targeting of DC has been

achieved by using antibodies directed against C-type lectins, such as DEC-205138,139 and DC-SIGN,133 which efficiently endocytose their cargo on binding. As molecular cloning of classic antibodies or fragments thereof offers serious challenges, alternatives have been explored. In members of the family of camilidae (i.e., Dromedary, Camel and Llama) a unique class of antibodies was described, which are only composed of two identical heavy chains as opposed to the conventional (four-chain) antibody repertoire.140 Thus, their antigen-binding part is composed of only one single immunoglobulin variable region (termed VHH, or Nanobody). These antigen-specific antibody fragments offer many advantages. They display a high solubility and the capacity to refold after denaturation although retaining their binding capacity. Cloning and selection of antigen-specific nanobodies obviate the need for construction and screening of large libraries, and for lengthy and unpredictable in vitro affinity maturation steps. Moreover, as nanobodies can be fused to other proteins, it should be possible to present them on the cell membrane of a producer cell line, such as HEK 293T, thus, generating lentiviral particles that incorporated a DC-specific nanobody in their envelope during budding as described above. This approach would combine the specificity of antibody targeting with the advantages of lentiviral vectors. All things considered, it remains to be seen whether specific targeting toward DC will be beneficial for lentivirus-based cancer immunotherapy, but the recent report by He et al.106 provides an important first clue for the feasibility of such an approach, as transduction of a specific subset of dermal DC seems to be responsible for the immune response. Nevertheless, as recent data showed that a viral infection promotes cross priming to DC, transduction of cells other than DC near the injection site might further improve subsequent processing and presentation of antigen by the DC.141 These issues need to be clarified in future by side-by-side comparisons of immunizations with broadly directed lentiviral vectors and vectors modified for specific transduction of DC.

855

Lentiviral vectors as a therapeutic tool: translation of preclinical animal models to the clinic Translating lentivirus-based animal cancer immunotherapy models to the clinic is challenging and requires a thorough evaluation of several issues: (1) dose toxicity studies, (2) biodistribution analyses and (3) testing of immunogenicity including therapeutic experiments. Furthermore, with regard to safety issues, it is critical to evaluate the occurrence of RCL and to strive for selective transduction of DC as discussed above. The main focus of the preclinical animal studies described in an earlier section of this paper was evaluating the immune response generated against the delivered transgene and testing the possibility of using TAA-encoding lentiviral vectors as a prophylactic and/ or therapeutic tool. In these studies, the potency of lentiviral vectors as anticancer vaccine has been repeatedly demonstrated. To get additional relevant information for human trials, an important issue that needs to be addressed in future animal studies is the in vivo biodistribution and Gene Therapy


856

replication of administered lentiviral vectors. Regarding biodistribution, several new tools for in vivo imaging in small animals have been described. For instance, lentiviruses encoding the sodium iodide importer or luciferase could be used for noninvasive gamma camera imaging and bioluminisence imaging, respectively. These tools will allow the evaluation of the optimal route of administration – footpad, intravenous, intradermal, subcutaneous or intramuscular injection – as well as the minimal dose required to transduce APC in situ, always aiming for an optimal antigen-specific immune response. To date, only a few publications have addressed biodistribution of lentiviral vectors on in vivo administration. The results of these studies suggest that depending on the route of administration, different DC subsets are selectively transduced in vivo. For instance, Esslinger et al.98 first evaluated tissue distribution after administration of a lentivector vaccine into the footpad of mice by a sensitive PCR assay, demonstrating transgene positivity at the injection site in lymph nodes and spleen. They further performed immunohistochemical analysis of the draining lymph nodes and spleen, demonstrating that the transgene positive cells are mainly DC. This was later extended by He et al.,106 who demonstrated that skin-derived DC (CD8/loCD11b+) are efficiently transduced on a single cutaneous injection of lentiviral vectors and that these DC subsequently migrate to the draining lymph nodes where they induce potent CTL responses. Another important issue that needs to be clarified before lentiviral vectors can be used as a clinical tool is dose toxicity. Here, the minimal dose required to transduce APC and still induce an effective antitumor immune response without significant side effects needs to be carefully determined.

Concerns in view of lentiviral vectors as anticancer vaccine Gene transfer vectors based on lentiviruses provide effective means for the delivery, integration and expression of transgenes in both dividing and nondividing mammalian cells. As mentioned earlier in this paper, many efforts have been undertaken to optimize the lentiviral vector system and this at the level of biosafety, good manufacturing practise (GMP) production, efficiency of gene transfer and gene expression. These efforts have led to the GMP production of lentiviral vectors devoid of RCL and producer cell contaminants, and to approval for use of lentiviral vectors in clinical applications. However, some concerns about biosafety of lentiviral vectors, mainly the generation of RCL and insertional mutagenesis remain. The risk of the generation of RCL through uncontrolled recombination in vector productions was largely addressed by engineering split plasmid systems for the generation of lentiviral particles, whereby a minimal overlap between the plasmids minimizes the risk of homologous recombination. So far, no RCL have been detected in virus preparations using highly sensitive techniques.142,143 In vivo, however, the integrated provirus could be rescued by recombination with wild-type retrovirus present in the patient (either HIV or some other virus that shares sequence homology to the provirus). The Gene Therapy

possibility that retroviral function could be provided in trans or that the vector genome could recombine with human endogenous retroviral sequences is an issue that will be difficult to assess in animal studies. Another risk related to such recombinations is the dissemination of new hybrid viruses from patients undergoing genetherapy treatment. Long-term monitoring of patients will be the most robust method for the detection of adverse events associated with lentivirus integration. With the recent reports of leukemia occurring in three out of ten SCID patients after treatment with CD34+ stem cells retrovirally transduced with the Yc gene,144 the risk of insertional mutagenesis with integrating vectors, including lentiviruses, is currently a major concern. Nevertheless, the comprehensive study by Dave et al.145 of 3000 insertions from nearly 1000 retrovirally induced murine hematopoietic tumors indicates that this particular case might be the result of an unfortunate conjunction of events, where the vector encoded Yc gene and the insertion site, the Lmo2 gene are collaborating oncogenes. Therefore, this turn of events should be highly exceptional as most therapeutic genes will not have oncogenic potential. It remains to be seen if lentiviral vectors will be safer than retroviral vectors, but continued efforts are being made to increase the safety of these vectors as well.146,147 A potential concern is that HIV-1 favors active genes for integration.148 Wildtype HIV, however, is not recognized to cause oncogenesis as superinfection with other viruses and loss of immune surveillance, not HIV itself, are responsible for malignancies observed in AIDS. Nevertheless, it has been described that delivery of nonprimate lentiviral vectors to fetal and neonatal mice leads to a high incidence of lentiviral vector-associated tumorigenesis.146 Therefore, efforts toward targeted integration in noncoding sequences as well as the development of nonintegrating lentiviral vectors are ongoing.149,150 With regard to targeted integration, the use of zinc fingers151 and the phage integrase phiC31152,153 have been described to mediate efficient, site-specific integration of target genes resulting in functional protein expression. To render the lentiviral vectors integration defective, mutations have been included in the integrase coding sequence.154 Recent, in vitro studies demonstrated that these integration-defective vectors mediate stable transduction.155,156 Furthermore, it has been shown in vivo in rodent ocular tissue that efficient and sustained transgene expression is obtained, resulting in substantial rescue of clinically relevant models of retinal degeneration, thus, demonstrating the potential of nonintegrating lentiviral vectors for therapeutic applications.149 However, all things considered, the use of lentiviral vectors for cancer immunotherapy holds less risk for oncogenesis. First, the level of transgene expression (and consequently the number of integrations) required to elicit an immune response is much lower than for obtaining therapeutic expression levels of a missing protein. As shown in Table 2 the doses used for immunization of mice with lentiviral vectors range from 1 106 to 2 107 TU, whereas the doses used for gene therapy range from 107 to 6 108 TU, which is about tenfold higher. Moreover, it has to be mentioned that for immunotherapy, the lentiviral vectors are systemically administered, whereas in the case of gene therapy, the lentiviral vectors are most often administered in the


857 Table 2 Overview of dose, route of administration and antigen used in the respective studies Reference

Dose

Route

Antigen

Esslinger et al.98 Palmowski et al.105 Kim et al.107 Rowe et al.110 Dullaers et al.104 He et al.106 Chapatte et al.108

2 107 EFU 106 PFU 1.6 106 PFU 107 IU 2 106/107 TU 106 TU 2 106 EFU

s.c. (footpad, tail base) i.v. (tail vein) s.c. (footpad) i.v. (tail vein) s.c. (footpad) s.c. (footpad) s.c. (tail base)

Cw3, mini Melan-A NY-ESO TRP-2 OVA OVA OVA Full-length Melan-A

Abbreviations: EFU, expression-forming units; PFU, plaque-forming units; TU, transducing units; i.v., intravenous; s.c., subcutaneous.

tissue of interest, resulting in transduction of these tissue-specific cells.157–161 Second, when using ex vivo-transduced DC or targeting DC in vivo, terminally differentiated, nondividing cells are transduced as opposed to extensively proliferating cells in the SCID trial, thereby posing less of a risk for oncogenic transformations. Finally, when an immune response is induced in vivo, the effector cells will most likely kill cells that express the target antigen, including the transduced APC, thus, further reducing the risk for oncogenesis. Even so, efforts to further improve the biosafety of lentiviral vectors should be continued.

Future perspectives Viral vectors based on retro- and lentiviral genomes will continue to play an important role as gene transfer reagents, because of their numerous advantages, among which (1) long-term expression, (2) low toxicity, (3) high capacity and (4) low anti-vector immunity allowing repeated administration. Besides expression of antigens for immunotherapeutic purposes, lentiviruses have other potential applications. They are very useful tools in the search for the function of newly identified molecules. Thus, the function of DC-related molecules that are picked up in microarrays can be investigated easily through either lentivirus-mediated overexpression or RNA interference through short hairpin RNA.162 Another application is the simultaneous modification of DC with antigen and immunomodulatory molecules via bicistronic vectors to influence the outcome of the immune response. Cotransduction with antigen and a costimulatory molecule could result in superactivated DC that are better stimulators than DC expressing the antigen alone. Lentiviral transduction of DC with CD40 ligand163 and gp34/OX40 ligand164 has already been shown to induce autonomous maturation and increased allostimulatory capacity, respectively. Similarly, cotransduction with antigen and a tolerogenic molecule, such as IL-10 or B7-H1, could result in antigen-specific tolerance, required in autoimmune diseases. Furthermore, the fact that direct injection of lentivirus reliably induces strong CTL responses against the transgene opens the possibility to exploit this technology for research applications other than those related to immunotherapy. Examples include the use of lentiviral vectors in combination with HLA transgenic mice as an efficient method for the identification of new epitopes that are also presented by human cells in vitro165 or for other applications where in vitro human systems are

inefficient or incomplete, such as the evaluation of the immune response in the presence or absence of certain components of the immune system, such as NK cells or Treg.

References 1 Ehrlich P. Ueber den jetzigen Stand der Karzinomforschung. Ned Tijdschr Geneesk 1909; 5: 273–290. 2 Burnet FM. The concept of immunological surveillance. Prog Exp Tumor Res 1970; 13: 1–27. 3 Boon T, van der Bruggen P. Human tumor antigens recognized by T lymphocytes. J Exp Med 1996; 183: 725–729. 4 Van den Eynde BJ, van der Bruggen P. T cell defined tumor antigens. Curr Opin Immunol 1997; 9: 684–693. 5 Lurquin C, Lethe B, De Plaen E, Corbiere V, Theate I, van Baren N et al. Contrasting frequencies of antitumor and anti-vaccine T cells in metastases of a melanoma patient vaccinated with a MAGE tumor antigen. J Exp Med 2005; 201: 249–257. 6 Yang Y, Huang CT, Huang X, Pardoll DM. Persistent Toll-like receptor signals are required for reversal of regulatory T cellmediated CD8 tolerance. Nat Immunol 2004; 5: 508–515. 7 Lang KS, Recher M, Junt T, Navarini AA, Harris NL, Freigang S et al. Toll-like receptor engagement converts T-cell autoreactivity into overt autoimmune disease. Nat Med 2005; 11: 138–145. 8 Fehervari Z, Sakaguchi S. CD4+ Tregs and immune control. J Clin Invest 2004; 114: 1209–1217. 9 Van Meirvenne S, Dullaers M, Heirman C, Straetman L, Michiels A, Thielemans K. In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells. Mol Ther 2005; 12: 922–932. 10 Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol 1999; 163: 5211–5218. 11 Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004; 10: 942–949. 12 Gautier G, Humbert M, Deauvieau F, Scuiller M, Hiscott J, Bates EE et al. A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J Exp Med 2005; 201: 1435–1446. 13 Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 2005; 6: 769–776. 14 Warger T, Osterloh P, Rechtsteiner G, Fassbender M, Heib V, Schmid B et al. Synergistic activation of dendritic cells by combined Toll-like receptor ligation induces superior CTL responses in vivo. Blood 2006; 108: 544–550. Gene Therapy


858 15 Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helperfree defective retrovirus. Cell 1983; 33: 153–159. 16 Vogt VM, Simon MN. Mass determination of rous sarcoma virus virions by scanning transmission electron microscopy. J Virol 1999; 73: 7050–7055. 17 Andreadis ST, Brott D, Fuller AO, Palsson BO. Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5*5 to 7*5 hours. J Virol 1997; 71: 7541–7548. 18 Le Doux JM, Davis HE, Morgan JR, Yarmush ML. Kinetics of retrovirus production and decay. Biotechnol Bioeng 1999; 63: 654–662. 19 Roe T, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 1993; 12: 2099–2108. 20 Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267. 21 Luciw P. Human Immunodeficiency Viruses and their Replication. Lippincot-Raven Publishers: Philadephia, 1996. 22 Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997; 15: 871–875. 23 Lewis PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68: 510–516. 24 Gallay P, Swingler S, Aiken C, Trono D. HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell 1995; 80: 379–388. 25 Gallay P, Swingler S, Song J, Bushman F, Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 1995; 83: 569–576. 26 Bukrinsky MI, Haggerty S, Dempsey MP, Sharova N, Adzhubel A, Spitz L et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 1993; 365: 666–669. 27 Heinzinger NK, Bukinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA et al. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994; 91: 7311–7315. 28 Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217–222. 29 Korin YD, Zack JA. Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J Virol 1998; 72: 3161–3168. 30 Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93: 11382–11388. 31 Gruber A, Kan-Mitchell J, Kuhen KL, Mukai T, Wong-Staal F. Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIVspecific cytotoxic T-lymphocyte response in vitro. Blood 2000; 96: 1327–1333. 32 Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463–8471. 33 Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157. 34 Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880. Gene Therapy

35 Ginn SL, Fleming J, Rowe PB, Alexander IE. Promoter interference mediated by the U3 region in early-generation HIV-1-derived lentivirus vectors can influence detection of transgene expression in a cell-type and species-specific manner. Hum Gene Ther 2003; 14: 1127–1137. 36 Sirven A, Pflumio F, Zennou V, Titeux M, Vainchenker W, Coulombel L et al. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 2000; 96: 4103–4110. 37 Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73: 2886–2892. 38 Sanders DA. No false start for novel pseudotyped vectors. Curr Opin Biotechnol 2002; 13: 437–442. 39 Schlegel A, Schaller J, Jentsch P, Kempf C. Semliki Forest virus core protein fragmentation: its possible role in nucleocapsid disassembly. Biosci Rep 1993; 13: 333–347. 40 Coil DA, Miller AD. Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus. J Virol 2004; 78: 10920–10926. 41 Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 1993; 90: 8033–8037. 42 Ory DS, Neugeboren BA, Mulligan RC. A stable humanderived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci USA 1996; 93: 11400–11406. 43 DePolo NJ, Reed JD, Sheridan PL, Townsend K, Sauter SL, Jolly DJ et al. VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol Ther 2000; 2: 218–222. 44 Croyle MA, Callahan SM, Auricchio A, Schumer G, Linse KD, Wilson JM et al. PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum. J Virol 2004; 78: 912–921. 45 Reiser J, Lai Z, Zhang XY, Brady RO. Development of multigene and regulated lentivirus vectors. J Virol 2000; 74: 10589–10599. 46 Kafri T, van Praag H, Gage FH, Verma IM. Lentiviral vectors: regulated gene expression. Mol Ther 2000; 1: 516–521. 47 Vigna E, Cavalieri S, Ailles L, Geuna M, Loew R, Bujard H et al. Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol Ther 2002; 5: 252–261. 48 Johansen J, Rosenblad C, Andsberg K, Moller A, Lundberg C, Bjorlund A et al. Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS. Gene Therapy 2002; 9: 1291–1301. 49 Georgievska B, Jakobsson J, Persson E, Ericson C, Kirik D, Lundberg C. Regulated delivery of glial cell line-derived neurotrophic factor into rat striatum, using a tetracyclinedependent lentiviral vector. Hum Gene Ther 2004; 15: 934–944. 50 Farson D, Witt R, McGuinness R, Dull T, Kelly M, Song J et al. A new-generation stable inducible packaging cell line for lentiviral vectors. Hum Gene Ther 2001; 12: 981–997. 51 Moreau-Gaudry F, Xia P, Jiang G, Perelman NP, Bauer G, Ellis J et al. High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors. Blood 2001; 98: 2664–2672. 52 De Palma M, Venneri MA, Naldini L. In vivo targeting of tumor endothelial cells by systemic delivery of lentiviral vectors. Hum Gene Ther 2003; 14: 1193–1206. 53 Lai Z, Brady RO. Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector. J Neurosci Res 2002; 67: 363–371.


859 54 Miyoshi H, Takahashi M, Gage FH, Verma IM. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA 1997; 94: 10319–10323. 55 Follenzi A, Sabatino G, Lombardo A, Boccaccio C, Naldini L. Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors. Hum Gene Ther 2002; 13: 243–260. 56 Oertel M, Rosencrantz R, Chen YQ, Thota PN, Sandhu JS, Dabeva MD et al. Repopulation of rat liver by fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors. Hepatology 2003; 37: 994–1005. 57 Uch R, Gerolami R, Faivre J, Hardwigsen J, Mathieu S, Mannoni P et al. Hepatoma cell-specific ganciclovir-mediated toxicity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences. Cancer Gene Ther 2003; 10: 689–695. 58 Yu D, Chen D, Chiu C, Razmazma B, Chow YH, Pang S. Prostate-specific targeting using PSA promoter-based lentiviral vectors. Cancer Gene Ther 2001; 8: 628–635. 59 Zheng JY, Chen D, Chan J, Yu D, Ko E, Pang S. Regression of prostate cancer xenografts by a lentiviral vector specifically expressing diphtheria toxin A. Cancer Gene Ther 2003; 10: 764–770. 60 Cui Y, Golob J, Kelleher E, Ye Z, Pardoll D, Cheng L. Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/ progenitor cells. Blood 2002; 99: 399–408. 61 Sinn PL, Sauter SL, McCray Jr PB. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors – design, biosafety, and production. Gene Therapy 2005; 12: 1089–1098. 62 Warnock J, Price T, Slade A, Al-Rubeai M. Use of a fluidizedbed bioreactor for the production of retroviral vectors for gene therapy. BioProcessing 2004; 3: 41–45. 63 Wu SC, Huang GY, Liu JH. Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture. Biotechnol Prog 2002; 18: 617–622. 64 Kafri T, van Praag H, Ouyang L, Gage FH, Verma IM. A packaging cell line for lentivirus vectors. J Virol 1999; 73: 576–584. 65 Klages N, Zufferey R, Trono D. A stable system for the hightiter production of multiply attenuated lentiviral vectors. Mol Ther 2000; 2: 170–176. 66 Xu K, Ma H, McCown TJ, Verma IM, Kafri T. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol Ther 2001; 3: 97–104. 67 Kuate S, Wagner R, Uberla K. Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors. J Gene Med 2002; 4: 347–355. 68 Ikeda Y, Takeuchi Y, Martin F, Cosset FL, Mitrophanous K, Collins M. Continuous high-titer HIV-1 vector production. Nat Biotechnol 2003; 21: 569–572. 69 Strang BL, Takeuchi Y, Relander T, Richter J, Bailey R, Sanders DA et al. Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells. J Virol 2005; 79: 1765–1771. 70 Bartz SR, Rogel ME, Emerman M. Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J Virol 1996; 70: 2324–2331. 71 Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 1995; 268: 429–431. 72 Miyazaki Y, Takamatsu T, Nosaka T, Fujita S, Martin TE, Hatanaka M. The cytotoxicity of human immunodeficiency

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

virus type 1 Rev: implications for its interaction with the nucleolar protein B23. Exp Cell Res 1995; 219: 93–101. Konvalinka J, Litterst MA, Welker R, Kottler H, Rippmann F, Heuser AM et al. An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity. J Virol 1995; 69: 7180–7186. Kumar M, Bradow BP, Zimmerberg J. Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64. Hum Gene Ther 2003; 14: 67–77. Sena-Esteves M, Tebbets JC, Steffens S, Crombleholme T, Flake AW. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 2004; 122: 131–139. Coleman JE, Huentelman MJ, Kasparov S, Metcalfe BL, Paton JF, Katovich MJ et al. Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol Genomics 2003; 12: 221–228. VandenDriessche T, Thorrez L, Naldini L, Follenzi A, Moons L, Berneman Z et al. Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigenpresenting cells in vivo. Blood 2002; 100: 813–822. Pham L, Ye H, Cosset FL, Russell SJ, Peng KW. Concentration of viral vectors by co-precipitation with calcium phosphate. J Gene Med 2001; 3: 188–194. Zhang B, Xia HQ, Cleghorn G, Gobe G, West M, Wei MQ. A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors. Gene Therapy 2001; 8: 1745–1751. Baekelandt V, Eggermont K, Michiels M, Nuttin B, Debyser Z. Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Therapy 2003; 10: 1933–1940. Scherr M, Battmer K, Eder M, Schule S, Hohenberg H, Ganser A et al. Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography. Gene Therapy 2002; 9: 1708–1714. Yamada K, McCarty DM, Madden VJ, Walsh CE. Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer. Biotechniques 2003; 34: 1074–1078, 1080. Schauber CA, Tuerk MJ, Pacheco CD, Escarpe PA, Veres G. Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro. Gene Therapy 2004; 11: 266–275. Scherr M, Battmer K, Blomer U, Ganser A, Grez M. Quantitative determination of lentiviral vector particle numbers by real-time PCR. Biotechniques 2001; 31: 520, 522, 524, passim. Sastry L, Johnson T, Hobson MJ, Smucker B, Cornetta K. Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Therapy 2002; 9: 1155–1162. Lizee G, Aerts JL, Gonzales MI, Chinnasamy N, Morgan RA, Topalian SL. Real-time quantitative reverse transcriptasepolymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 2003; 14: 497–507. Logan AC, Nightingale SJ, Haas DL, Cho GJ, Pepper KA, Kohn DB. Factors influencing the titer and infectivity of lentiviral vectors. Hum Gene Ther 2004; 15: 976–988. Breckpot K, Dullaers M, Bonehill A, van Meirvenne S, Heirman C, de Greef C et al. Lentivirally transduced dendritic cells as a tool for cancer immunotherapy. J Gene Med 2003; 5: 654–667. Unutmaz D, KewalRamani VN, Marmon S, Littman DR. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J Exp Med 1999; 189: 1735–1746. Gene Therapy


860 90 Schroers R, Sinha I, Segall H, Schmidt-Wolf IG, Rooney CM, Brenner MK et al. Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system. Mol Ther 2000; 1: 171–179. 91 Dyall J, Latouche JB, Schnell S, Sadelain M. Lentivirustransduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes. Blood 2001; 97: 114–121. 92 Firat H, Zennou V, Garcia-Pons F, Ginhoux F, Cochet M, Danos O et al. Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy. J Gene Med 2002; 4: 38–45. 93 Lizee G, Gonzales MI, Topalian SL. Lentivirus vector-mediated expression of tumor-associated epitopes by human antigen presenting cells. Hum Gene Ther 2004; 15: 393–404. 94 Oki M, Ando K, Hagihara M, Miyatake H, Shimizu T, Miyoshi H et al. Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells. Exp Hematol 2001; 29: 1210–1217. 95 Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH, Trono D. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 2000; 96: 3392–3398. 96 Sumimoto H, Tsuji T, Miyoshi H, Hagihara M, TakadaYamazaki R, Okamoto S et al. Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells. J Immunol Methods 2002; 271: 153–165. 97 Metharom P, Ellem KA, Schmidt C, Wei MQ. Lentiviral vectormediated tyrosinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma. Hum Gene Ther 2001; 12: 2203–2213. 98 Esslinger C, Chapatte L, Finke D, Miconnet I, Guillaume P, Levy F et al. In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+) T cell responses. J Clin Invest 2003; 111: 1673–1681. 99 Zarei S, Leuba F, Arrighi JF, Hauser C, Piguet V. Transduction of dendritic cells by antigen-encoding lentiviral vectors permits antigen processing and MHC class I-dependent presentation. J Allergy Clin Immunol 2002; 109: 988–994. 100 Haas DL, Case SS, Crooks GM, Kohn DB. Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. Mol Ther 2000; 2: 71–80. 101 Dullaers M, Breckpot K, Van Meirvenne S, Bonehill A, Tuyaerts S, Michiels A et al. Side-by-side comparison of lentivirally transduced and mRNA-electroporated dendritic cells: implications for cancer immunotherapy protocols. Mol Ther 2004; 10: 768–779. 102 Breckpot K, Heirman C, De Greef C, van der Bruggen P, Thielemans K. Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses. J Immunol 2004; 172: 2232–2237. 103 He Y, Zhang J, Mi Z, Robbins P, Falo Jr LD. Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity. J Immunol 2005; 174: 3808–3817. 104 Dullaers M, Van Meirvenne S, Heirman C, Straetman L, Bonehill A, Aerts JL et al. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors. Gene Therapy 2006; 13: 630–640. 105 Palmowski MJ, Lopes L, Ikeda Y, Salio M, Cerundolo V, Collins MK. Intravenous injection of a lentiviral vector encoding NYESO-1 induces an effective CTL response. J Immunol 2004; 172: 1582–1587. 106 He Y, Zhang J, Donahue C, Falo Jr LD. Skin-derived dendritic cells induce potent CD8(+) T cell immunity in recombinant Gene Therapy

107

108

109

110

111

112

113

114

115

116

117

118

119

120 121 122

123

124

lentivector-mediated genetic immunization. Immunity 2006; 24: 643–656. Kim JH, Majumder N, Lin H, Watkins S, Falo Jr LD, You Z. Induction of therapeutic antitumor immunity by in vivo administration of a lentiviral vaccine. Hum Gene Ther 2005; 16: 1255–1266. Chapatte L, Colombetti S, Cerottini JC, Levy F. Efficient induction of tumor antigen-specific CD8+ memory T cells by recombinant lentivectors. Cancer Res 2006; 66: 1155–1160. Rosenberg SA, Sherry RM, Morton KE, Scharfman WJ, Yang JC, Topalian SL et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J Immunol 2005; 175: 6169–6176. Rowe HM, Lopes L, Ikeda Y, Bailey R, Barde I, Zenke M et al. Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene. Mol Ther 2006; 13: 310–319. McMichael AJ, Phillips RE. Escape of human immunodeficiency virus from immune control. Annu Rev Immunol 1997; 15: 271–296. Liu SL, Schacker T, Musey L, Shriner D, McElrath MJ, Corey L et al. Divergent patterns of progression to AIDS after infection from the same source: human immunodeficiency virus type 1 evolution and antiviral responses. J Virol 1997; 71: 4284–4295. Cao Y, Qin L, Zhang L, Safrit J, Ho DD. Characterization of long-term survivors of human immunodeficiency virus type 1 infection. Immunol Lett 1996; 51: 7–13. Fonteneau JF, Larsson M, Beignon AS, McKenna K, Dasilva I, Amara A et al. Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells. J Virol 2004; 78: 5223–5232. Beignon AS, McKenna K, Skoberne M, Manches O, DaSilva I, Kavanagh DG et al. Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions. J Clin Invest 2005; 115: 3265–3275. Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, Bazan F et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 2001; 194: 863–869. Tan PH, Beutelspacher SC, Xue SA, Wang YH, Mitchell P, McAlister JC et al. Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy. Blood 2005; 105: 3824–3832. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem 1998; 67: 227–264. Taylor SS, Haste NM, Ghosh G. PKR and eIF2alpha: integration of kinase dimerization, activation, and substrate docking. Cell 2005; 122: 823–825. Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol 2006; 7: 131–137. Kapsenberg ML. Dendritic-cell control of pathogen-driven Tcell polarization. Nat Rev Immunol 2003; 3: 984–993. Brown BD, Sitia G, Annoni A, Hauben E, Sergi Sergi L, Zingale A et al. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance. Blood 2006 [Epub ahead of print]. Zhang H, Dornadula G, Pomerantz RJ. Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells. J Virol 1996; 70: 2809–2824. Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F et al. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat Immunol 2006; 7: 40–48.


861 125 Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 2006; 24: 93–103. 126 Pichlmair A, Diebold SS, Gschmeissner S, Takeuchi Y, Ikeda Y, Collins MK et al. Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via tolllike receptor 9. J Virol 2007; 81: 539–547. 127 Buller RM, Holmes KL, Hugin A, Frederickson TN, Morse III HC. Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells. Nature 1987; 328: 77–79. 128 Shedlock DJ, Shen H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 2003; 300: 337–339. 129 Sun JC, Bevan MJ. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 2003; 300: 339–342. 130 Janssen EM, Lemmens EE, Wolfe T, Christen U, von Herrath MG, Schoenberger SP. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 2003; 421: 852–856. 131 Marzo AL, Vezys V, Klonowski KD, Lee SJ, Muralimohan G, Moore M et al. Fully functional memory CD8 T cells in the absence of CD4 T cells. J Immunol 2004; 173: 969–975. 132 Noti JD, Reinemann BC, Petrus MN. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription. Mol Cell Biol 1996; 16: 2940–2950. 133 Dzionek A, Sohma Y, Nagafune J, Cella M, Colonna M, Facchetti F et al. BDCA-2, a novel plasmacytoid dendritic cellspecific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction. J Exp Med 2001; 194: 1823–1834. 134 Takahara K, Omatsu Y, Yashima Y, Maeda Y, Tanaka S, Iyoda T et al. Identification and expression of mouse Langerin (CD207) in dendritic cells. Int Immunol 2002; 14: 433–444. 135 Chandrashekran A, Gordon MY, Casimir C. Targeted retroviral transduction of c-kit+ hematopoietic cells using novel ligand display technology. Blood 2004; 104: 2697–2703. 136 Chandrashekran A, Gordon MY, Darling D, Farzaneh F, Casimir C. Growth factor displayed on the surface of retroviral particles without manipulation of envelope proteins is biologically active and can enhance transduction. J Gene Med 2004; 6: 1189–1196. 137 Yang L, Bailey L, Baltimore D, Wang P. Targeting lentiviral vectors to specific cell types in vivo. Proc Natl Acad Sci USA 2006; 103: 11479–11484. 138 Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med 2002; 196: 1627–1638. 139 Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii S, Soares H et al. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J Exp Med 2004; 199: 815–824. 140 Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB et al. Naturally occurring antibodies devoid of light chains. Nature 1993; 363: 446–448. 141 Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, Alexopoulou L et al. Toll-like receptor 3 promotes crosspriming to virus-infected cells. Nature 2005; 433: 887–892. 142 Segall H, Sutton RE. Detection of replication-competent lentiviral particles. Methods Mol Biol 2003; 229: 87–94. 143 Escarpe P, Zayek N, Chin P, Borellini F, Zufferey R, Veres G et al. Development of a sensitive assay for detection of replicationcompetent recombinant lentivirus in large-scale HIV-based vector preparations. Mol Ther 2003; 8: 332–341.

144 Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419. 145 Dave UP, Jenkins NA, Copeland NG. Gene therapy insertional mutagenesis insights. Science 2004; 303: 333. 146 Trono D. Virology. Picking the right spot. Science 2003; 300: 1670–1671. 147 Sadelain M. Insertional oncogenesis in gene therapy: how much of a risk? Gene Therapy 2004; 11: 569–573. 148 Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 2002; 110: 521–529. 149 Yanez-Munoz RJ, Balaggan KS, MacNeil A, Howe SJ, Schmidt M, Smith AJ et al. Effective gene therapy with nonintegrating lentiviral vectors. Nat Med 2006; 12: 348–353. 150 Bushman F. Targeting retroviral integration? Mol Ther 2002; 6: 570–571. 151 Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435: 646–651. 152 Groth AC, Olivares EC, Thyagarajan B, Calos MP. A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 2000; 97: 5995–6000. 153 Held PK, Olivares EC, Aguilar CP, Finegold M, Calos MP, Grompe M. In vivo correction of murine hereditary tyrosinemia type I by phiC31 integrase-mediated gene delivery. Mol Ther 2005; 11: 399–408. 154 Engelman A. In vivo analysis of retroviral integrase structure and function. Adv Virus Res 1999; 52: 411–426. 155 Vargas Jr J, Gusella GL, Najfeld V, Klotman ME, Cara A. Novel integrase-defective lentiviral episomal vectors for gene transfer. Hum Gene Ther 2004; 15: 361–372. 156 Saenz DT, Loewen N, Peretz M, Whitwam T, Barraza R, Howell KG et al. Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants. J Virol 2004; 78: 2906–2920. 157 Yoshimitsu M, Higuchi K, Dawood F, Rasaiah VI, Ayach B, Chen M et al. Correction of cardiac abnormalities in fabry mice by direct intraventricular injection of a recombinant lentiviral vector that engineers expression of alpha-galactosidase A. Circ J 2006; 70: 1503–1508. 158 Carbonaro DA, Jin X, Petersen D, Wang X, Dorey F, Kil KS et al. In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency. Mol Ther 2006; 13: 1110–1120. 159 Di Domenico C, Di Napoli D, Gonzalez YRE, Lombardo A, Naldini L, Di Natale P. Limited transgene immune response and long-term expression of human alpha-L-iduronidase in young adult mice with mucopolysaccharidosis type I by liverdirected gene therapy. Hum Gene Ther 2006; 17: 1112–1121. 160 Vercammen L, Van der Perren A, Vaudano E, Gijsbers R, Debyser Z, Van den Haute C et al. Parkin protects against neurotoxicity in the 6-hydroxydopamine rat model for Parkinson’s disease. Mol Ther 2006; 14: 716–723. 161 Wazen RM, Moffatt P, Zalzal SF, Daniel NG, Westerman KA, Nanci A. Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector. Gene Therapy 2006; 13: 1595–1602. 162 Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003; 33: 401–406. 163 Koya RC, Kasahara N, Favaro PM, Lau R, Ta HQ, Weber JS et al. Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery. J Immunother 2003; 26: 451–460. Gene Therapy


862 164 Kobayashi M, Takaori-Kondo A, Fukunaga K, Miyoshi H, Uchiyama T. Lentiviral gp34/OX40L gene transfer into dendritic cells facilitates alloreactive CD4+ T-cell response in vitro. Int J Hematol 2004; 79: 377–383. 165 Rohrlich PS, Cardinaud S, Lule J, Montero-Julian FA, Prodhomme V, Firat H et al. Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells. Hum Immunol 2004; 65: 514–522. 166 Delenda C. Lentiviral vectors: optimization of packaging, transduction and gene expression. J Gene Med 2004; 6 (Suppl 1): S125–S138. 167 Poeschla EM, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 1998; 4: 354–357. 168 Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M. Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proc Natl Acad Sci USA 1996; 93: 15266–15271. 169 Mochizuki H, Schwartz JP, Tanaka K, Brady RO, Reiser J. Hightiter human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J Virol 1998; 72: 8873–8883. 170 Wong LF, Azzouz M, Walmsley LE, Askham Z, Wilkes FJ, Mitrophanous KA et al. Transduction patterns of pseudotyped lentiviral vectors in the nervous system. Mol Ther 2004; 9: 101–111. 171 Beyer WR, Westphal M, Ostertag W, von Laer D. Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. J Virol 2002; 76: 1488–1495. 172 Christodoulopoulos I, Cannon PM. Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that

Gene Therapy

173

174

175

176

177

178

179

180

prevent its incorporation into lentivirus vectors. J Virol 2001; 75: 4129–4138. Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, McKeating JA. CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol 2004; 78: 1448–1455. Flint M, Logvinoff C, Rice CM, McKeating JA. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins. J Virol 2004; 78: 6875–6882. Kowolik CM, Yee JK. Preferential transduction of human hepatocytes with lentiviral vectors pseudotyped by Sendai virus F protein. Mol Ther 2002; 5: 762–769. Marzi A, Gramberg T, Simmons G, Moller P, Rennekamp AJ, Krumbiegel M et al. DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J Virol 2004; 78: 12090–12095. Kobinger GP, Weiner DJ, Yu QC, Wilson JM. Filoviruspseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 2001; 19: 225–230. Liu SL, Halbert CL, Miller AD. Jaagsiekte sheep retrovirus envelope efficiently pseudotypes human immunodeficiency virus type 1-based lentiviral vectors. J Virol 2004; 78: 2642–2647. Sinn PL, Penisten AK, Burnight ER, Hickey MA, Williams G, McCoy DM et al. Gene transfer to respiratory epithelia with lentivirus pseudotyped with Jaagsiekte sheep retrovirus envelope glycoprotein. Hum Gene Ther 2005; 16: 479–488. Stitz J, Buchholz CJ, Engelstadter M, Uckert W, Bloemer U, Schmitt I et al. Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10A1. Virology 2000; 273: 16–20.