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At least 19 recordsLinked to original sources
PubMed2002

Development of gene therapy for hematopoietic stem cells using lentiviral vectors.

Lentiviral vectors are promising tools for the development of gene therapy since they can transduce both quiescent and dividing target cells. Lentiviral vectors may be particularly promising gene delivery tools for hematopoietic stem cells since these target cells tend to be quiescent and are therefore difficult target cells for vectors that require dividing targets. Human hematopoietic stem cells that can repopulate NOD/SCID mice have been efficiently transduced using HIV-1-based lentiviral vectors and similar vectors can also transduce murine hematopoietic stem cells. HIV-1 vectors that contain strong general promoters can generate high levels of transgene expression and very high expression levels can be generated in erythroid cells in vivo using beta-globin regulatory sequences to control the expression of the transgene. Current lentiviral vectors have a similar level of biosafety as oncoretroviral vectors and can therefore theoretically be used in clinical gene therapy protocols. Future challenges include the generation of lentiviral vectors that can express more than one transgene at high levels and the generation of safe permanent packaging cells for practical use in clinical gene therapy trials.

Animals↗
PubMed2006

Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors.

Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped lentiviral vectors (RRV-LV)). The RRV is an alphavirus with an extremely broad host range, including the cells of the central nervous system. Previous studies have shown that lentiviral vectors can be efficiently pseudotyped with this envelope protein and have demonstrated promising features of such vectors, including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells.

Animals↗
PubMed2000

Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors.

Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.

Antigens, CD34↗
PubMed2001

Functional correction of fanconi anemia group C hematopoietic cells by the use of a novel lentiviral vector.

Lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. Here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV). EIAV is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. We employed the EIAV vector system to test for gene transfer to human Fanconi anemia (FA) hematopoietic cells by comparison with HIV-1- and Moloney murine leukemia virus-based systems. Fanconi anemia is characterized by bone marrow failure secondary to stem cell dysfunction. Fanconi anemia group C EBV-transformed lymphoblasts were transduced with all three viral vectors. Phenotypic correction of FA cells, as measured by mitomycin C drug resistance, was observed with a similar efficiency in all vector systems. This is the first description of lentiviral correction of FA cells and suggests that lentiviral vectors may be useful for FA gene transfer.

Cell Cycle↗
PubMed2004

Factors influencing the titer and infectivity of lentiviral vectors.

Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34+ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G glycoprotein (VSV-G).

Antigens, CD34↗
PubMed2001

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors.

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.

Animals↗
PubMed2006

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

Biological Assay↗
PubMed2001

A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors.

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50,000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10,000 g for 2 h at 4 degrees C. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3,000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10,000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Animals↗
PubMed2000

A stable system for the high-titer production of multiply attenuated lentiviral vectors.

Lentiviral vectors open exciting perspectives for the genetic treatment of a wide array of inherited and acquired diseases, owing to their ability to govern the efficient delivery, integration, and long-term expression of transgenes into nondividing cells both in vitro and in vivo. The genomic complexity of HIV, where a whole set of genes encode virulence factors essential for pathogenesis but not required for gene transfer, allowed a major step toward clinical acceptability through the creation of multiply attenuated packaging systems. Until now, however, vector particles could only be produced by transient transfection because no high-output, stable packaging cell line was available that produced the latest generation of HIV-based vectors. Here we describe such a line, based on the doxycycline-repressible expression of HIV-1 Rev/Gag/Pol and of the vesicular stomatitis virus G envelope (VSV G) in 293 human embryonic kidney cells. Upon induction, the LVG clones can produce 1 to 20 HeLa-transducing units per cell per day for about a week, a yield that compares favorably with that of transiently transfected 293T cells. These virions exhibit functional properties similar to those of viruses produced transiently, in particular the ability to transduce nonmitotic targets. This system will facilitate the further development of lentiviral vectors for gene therapy.

Blotting, Western↗
PubMed2004

Transactivator and structurally optimized inducible lentiviral vectors.

Lentiviral vectors offer well-recognized advantages as a gene delivery system both for the analysis of gene function and as a vehicle for gene therapy. In the present study optimized HIV-1-based vector systems that display efficient doxycycline (Dox)-dependent transgene expression in vitro and in vivo have been developed through the modification of factors that contribute to basal activity levels. Dissection of HIV-1 vectors harboring a tTA-dependent transgene expression cassette revealed several mechanisms that account for Dox-independent transgene expression, including those mediated by an internal CMV promoter, as well as a potential contribution from fusion proteins generated by translational readthrough. A precipitous reduction in basal activity levels was accomplished by separating the transactivator and the transgene cassettes into a binary vector system and by relocating the inducible promoter to the U3 region of the LTR. In addition, substituting the VP16 portion of tTA with the human p65 transactivating domain improved Dox-dependent transgene expression in a number of cell types. Optimizing HIV-1-based vectors culminated in a "toolbox" of vectors suitable for transgene delivery in vitro and in vivo, as conveyed by our ability to control the Dox-dependent differentiation of embryonic fibroblasts into muscle cells in vitro and transgene expression in rat brains.

Animals↗
PubMed2003

Efficient production of human FVIII in hemophilic mice using lentiviral vectors.

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken beta-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.

Animals↗
PubMed2000

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors.

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.

Alu Elements↗
PubMed2003

Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy.

Lentiviral vectors have become attractive delivery vehicles for gene therapy investigators. Specifically, the ability of lentiviral vectors to integrate into nondividing cells and provide stable and long-term gene expression in vivo is a desirable attribute of gene therapy approaches. We report here a simple method for generating transgenic mice using lentiviral vectors, which could be useful models for gene therapy. After removal of the zona pellucida, fertilized eggs were co-incubated with oncoretroviral or lentiviral vectors. The resulting blastocysts were transferred into uteri of pseudo-pregnant females. In both cases, around 60-70% of founder pups were transgenic as determined by PCR analysis. Southern blot analysis revealed that the transgenes were integrated at different genetic loci and transmitted through the germ line. Most of the transgenes delivered by lentiviral vectors were expressed in transgenic mice, although those delivered by oncoretroviral vectors were completely silenced. When the upstream sequences of the rhodopsin gene and the red pigment gene were used as tissue-specific promoters, consistent enhanced green fluorescent protein (EGFP) expression was observed in rod and cone photoreceptor cells, respectively, in retina. However, mice generated with the corneal epithelium-specific keratin-12 promoter displayed EGFP expression not only in cornea but also in other tissues of the mouse. We conclude that the generation of transgenic mice using lentiviral vectors is a simple and robust method to evaluate the promoter specificity in lentiviral vectors in vivo prior to undertaking a gene therapy strategy.

Animals↗
PubMed2003

Multiply attenuated, self-inactivating lentiviral vectors efficiently deliver and express genes for extended periods of time in adult rat cardiomyocytes in vivo.

BACKGROUND: Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. METHODS AND RESULTS: Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1alpha (EF-1alpha) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, approximately 5). The CMV promoter achieved higher EGFP expression levels than the EF-1alpha and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by approximately 2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by approximately 4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by approximately 7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1alpha promoter-containing lentivectors. Peak EGFP expression with titer-matched adenovectors was approximately 35% higher than with CMV lentivectors but was lost rapidly over time. CONCLUSIONS: Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.

Adenoviridae↗
PubMed2003

Regulating gene expression using self-inactivating lentiviral vectors containing the mifepristone-inducible system.

Methods to regulate gene expression in vitro and in vivo are currently areas of intense research. The present study, therefore, was designed to determine the efficacy of transgene expression using the GeneSwitch mifepristone-regulatable system within the context of an integrating HIV-1 vector. Lentiviral transfer plasmids expressing the red (DsRed2) and green fluorescent protein (EGFP) markers were constructed for in vitro assessment on the basal and mifepristone-induced cell activation levels by FACS analyses. In our design, efficient cell activation and transgene expression were found using a binary lentivector system i.e., the trans-activator, Switch, and the inducible promoter-transgene expression cassette were cloned into separate vectors. Note that the Switch trans-activator performed optimally when cloned into the reverse-orientation, but the inducible promoter containing lentivector did not appear to be dependent upon the orientation within the lentivector backbone. This binary lentivector system resulted in tightly regulated transgene expression, with low basal cell activation in the absence of mifepristone (MFP). Upon induction, a 41- to 275-fold increase in the number of DsRed2- and EGFP-positive cells were detected (n=3). To determine the inducing ability of the GeneSwitch, we cloned the human alpha(1)-antitrypsin cDNA into the optimal lentiviral vector and transduced HeLa and Huh7 cells at increasing lentivector doses as determined by p24 Gag ELISA. We found that MFP could induce the expression of hAAT protein in HeLa cells from 310 to 15,000 ng hAAT/10(6) cells/24 h, which was a 48-fold induction. Similar results were observed in huH7 cells. In all, this study demonstrates that the GeneSwitch system can be designed within the context of a lentiviral vector for in vitro gene transfer, and this may also provide a viable method for temporally regulating gene expression for therapeutic applications in vivo or ex vivo.

Cell Line↗
PubMed2006

Transient gene expression by nonintegrating lentiviral vectors.

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

Antigens, CD34↗
PubMed2001

Analysis of lenti- and trans-lentiviral vector genetic recombination.

Lentiviral vectors hold great promise for gene therapy, and clinical trials to examine their safety and efficacy for treating human disease are being planned. The principle concern for safety is that genetic recombination among components of the vector could lead to the emergence of replication competent retrovirus (RCR). Using a sensitive method for detecting genetic recombination, we found that the current design of lentiviral vectors permits the generation of envelope-deficient recombinant lentivirus, stable integration of the recombinant into chromosomes of transduced cells, and mobilization of the recombinant genomes to other cells when pseudotyped with an exogenous envelope. We split the lentiviral packaging construct (Gag/Gag-Pol) into two separate parts: one that expresses Gag and Gag-Pro, and another that expresses Pol (reverse transcriptase [RT] and integrase [IN]) as a fusion partner of Vpr (Vpr-RT-IN). This "trans-lentiviral" vector efficiently transduces non-dividing cells and achieves titres greater than 10(6) U/ml or 10(8) IU/ml after concentration by ultracentrifugation. The trans-lentiviral vector disarms the Gag-Pol structure and prevents the generation of recombinants containing functional RT and IN. Since RT and IN are absolutely required for any type of RCR and DNA mobilization, this new class of lentiviral vector, in combination with our sensitive in vitro assay for monitoring regeneration of the gag-pol structure, offers a unique advantage for predicting vector safety for clinical applications.

Base Sequence↗
PubMed2006

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors.

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Animals↗