Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.     

The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo

Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.     

人Hes1-shRNA，Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的构建人Hesl-shRNA和Hes5-shRNA慢病毒表达载体,为Notch—Hes信号通路的相关研究奠定基础。方法根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR／U6入门载体。通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列。将含有效干扰序列的入门载体与pLenti6／BLOCK—iT—DEST载体进行LR重组构建Hesl—shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒。用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达。结果分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对HeM,Hes5蛋白的表达有显著抑制作用。结论成功构建了Hesl—shRNA和Hes5-shRNA慢病毒表达载体。     

Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.     

Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.     

Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity

Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-gamma by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.     

Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles

Transgenic HIV-1-derived lentiviral particles are at the forefront of current gene therapy and tissue engineering initiatives, which will require optimal protocols for large-scale production of clinical-grade therapeutic lentiviruses. Production of latest-generation self-inactivating lentiviral particles requires cotransfection of mammalian production cell lines with two helper plasmids along with the lentivector, whose transgene-encoding expression cassette is the only genetic information stably transduced into target chromosomes. Capitalizing on a recently designed lentiviral expression vector family, we conducted rigorous analysis of production-relevant parameters including transfection, cell density, media composition, temperature, relative (helper) vector concentrations and genetic configuration. Comparative analysis of lentiviral particle performance (VP) was based on the viral titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of lentiviral particles produced (correlating with p24 production levels) (VP=titer/viral particle number). Optimal lentiviral production parameters, resulting in up to 132-fold greater VP compared to standard protocols, required (i) CaPO4-based transfection (ii) of helper plasmids and lentivector at a fixed concentration ratio (helper plasmid I:helper plasmid II:lentivector=1:1:2) (iii) into 1x10(5) human embryonic kidney cells/cm2 (HEK293-T) (iv) cultivated at 37 degrees C (v) in Advanced D-MEM medium supplemented with (vi) 2% fetal calf serum, (vii) and a culture additive containing 0.01 mM cholesterol, 0.01 mM egg's lecithin and 1x chemically defined lipid concentrate. (viii) Furthermore, constitutive transgene expression units placed in a forward polyadenylation site (pA)-free orientation relative to the lentivector backbone resulted in optimal transgene transduction/expression. Our studies suggest that detailed knowledge of lentivector design and the production of lentiviral particles will advance large-scale manufacturing of clinically relevant lentiviruses for future gene therapy applications.     

Efficient gene targeting mediated by a lentiviral vector-associated meganuclease

Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes.     

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.     

Dendritic Cell-Specific Delivery of Flt3L by Coronavirus Vectors Secures Induction of Therapeutic Antitumor Immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8⁺ T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8⁺ T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity.     

Lentiviral vectors: basic to translational

More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput '-omics' era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.     

A model vaccine exploiting fungal mannosylation to increase antigen immunogenicity

Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4+ T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because macrophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glycosylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity.     

Conventional dendritic cells are required for the activation of helper-dependent CD8 T cell responses to a model antigen after cutaneous vaccination with lentiviral vectors

Cutaneous vaccination with lentiviral vectors generates systemic CD8 T cell responses that have the potential to eradicate tumors for cancer immunotherapy. However, although s.c. immunization with <1 million lentiviral particles clearly primes cytotoxic T cells, vaccination with much higher doses has routinely been used to define the mechanisms of T cell activation by lentiviral vectors. In particular, experiments to test presentation of lentiviral Ags by dendritic cells (DC) require injection of high viral titers, which may result in aberrant transduction of different DC populations. We exploited inducible murine models of DC depletion to investigate which DC prime the lentiviral response after s.c. immunization with low doses of lentiviral particles. In this article, we demonstrate that conventional DC are required to present Ag to CD8 T cells in draining lymph nodes. Langerhans cells are not required to activate the effector response, and neither Langerhans cells nor plasmacytoid DC are sufficient to prime Ag-specific T cells. Immunization drives the generation of endogenous long-lived memory T cells that can be reactivated to kill Ag-specific targets in the absence of inflammatory challenge. Furthermore, lentiviral vaccination activates expansion of endogenous CD4 Th cells, which are required for the generation of effector CD8 T cells that produce IFN-γ and kill Ag-specific targets. Collectively, we demonstrate that after cutaneous immunization with lentiviral particles, CD4-licensed lymph node conventional DC present Ag to CD8 T cells, resulting in the generation of protective endogenous antitumor immunity that may be effective for cancer immunotherapy.     

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination

Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and induce antigen expression, thus providing an efficient method for delivering DC-directed vaccines. In this study, we constructed a stable producer line (LV-MGFP) for synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent protein (GFP) by a concatemeric array transfection technique. We demonstrated that the established stable clones could routinely produce vector supernatants with titers above 10(7) transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. The producer cells were also capable of generating similar titers of DC-LVs in serum-free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the producer cells to manufacture DC-LVs with both improved titers and enhanced potency to evoke antigen-specific CD8(+) T cell responses in mice. The stable lines could accommodate the replacement of the internal murine stem cell virus (MSCV) promoter with the human ubiquitin-C (Ubi) promoter in the lentiviral backbone. The resulting DC-LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity. Based on accumulated evidence, our studies support the application of this production method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based immunization.