Efficient Gene Transfer to Human Peripheral Blood Monocyte-Derived Dendritic Cells Using Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors

Abstract

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.

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination

“…The titer of the viral preparations and the presence of replication-competent lentivirus (RCL) were determined as described previously. 34,59 No RCL was detectable in any of the vector preparations used in this study.…”

“…7,9,10,[31][32][33] We, and others, have shown the potential of HIV-1-based lentiviral vectors in delivering transgene to human monocyte-derived DCs and the induction of T-cell responses. 25,[34][35][36] The initial aim of the current study was to develop and optimize a clinically feasible method to generate gene-modified DC vaccines using an SIN lentiviral vector for cancer immunotherapy. Similar to previous findings, 37,38 the current study demonstrate that the SIN lentiviral vector could reproducibly transduce PB monocyte-derived immature DCs, resulting in the high level of transgene expression.…”

“…The lentiviral vector particles were produced in 293T cells (American Type Culture Collection, Manassas, VA, USA) by three-plasmid transfection using a calcium phosphate transfection kit (Invitrogen) as described previously, 34,59 except that Opti-MEM-1 containing 5% HS was used as culture medium. The viral supernatants were concentrated to 50 Â by ultracentrifugation (50 000 g, 1.5 h, 41C).…”

Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro

“…Unfortunately, VSV-G-pseudotyped lentivectors can efficiently transduce APCs from mice and humans, and can elicit immune responses against the transgene product, thus potentially negating the therapeutic effects of the protein expression. [7][8][9][10] Systemic administration of viral vectors also exposes the particles to possible inactivation by serum complement. 11 Complement inactivation of vectors is dependent on the species derivation of the cell line used to produce the vectors, as well as the identity of the envelope glycoprotein used for pseudotyping.…”

Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense

“…[1][2][3][4][5][6][7][8] The use of HIV-based conditionally replicating vectors expressing anti-HIV genes for the treatment of AIDS offers several theoretical advantages over the MoMLVbased vector systems. Unlike MoMLV, lentiviral vectors can effectively transduce both dividing and non-dividing cells such as resting T cells, macrophages or dendritic cells, 9,10 which may play an important role in the maintenance of HIV-1 infection. 11,12 In addition to the effect provided by the expression of an anti-HIV gene, these vectors can inhibit HIV-1 replication by sequestering the HIV-1 regulatory proteins Tat and Rev, by interfering with reverse transcription and/or by competing for packaging into HIV-1 encoded virions, facilitating the spread of the vector to unprotected cells in vivo.…”