title: Large-scale feasibility of gene transduction into human CD34+ cell-derived dendritic cells by adenoviral/polycation complex.
abstract: With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.
authors.affiliation: Division of Medical Oncology, Istituto Nazionale Tumori, Milan, Italy. dinicola@istitutotumori.mi.it
authors.forename: M
authors.initials: M
authors.lastname: Di Nicola
authors.forename: C
authors.initials: C
authors.lastname: Carlo-Stella
authors.forename: M
authors.initials: M
authors.lastname: Milanesi
authors.forename: M
authors.initials: M
authors.lastname: Magni
authors.forename: P
authors.initials: P
authors.lastname: Longoni
authors.forename: R
authors.initials: R
authors.lastname: Mortarini
authors.forename: A
authors.initials: A
authors.lastname: Anichini
authors.forename: R
authors.initials: R
authors.lastname: Tomanin
authors.forename: M
authors.initials: M
authors.lastname: Scarpa
authors.forename: A M
authors.initials: AM
authors.lastname: Gianni
mesh_terms: D000256:Adenoviridae; D000469:Alkaline Phosphatase / Q000235:genetics; D018952:Antigens, CD34; D019496:Cancer Vaccines; D002411:Cation Exchange Resins; D002412:Cations; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D003713:Dendritic Cells; D005240:Feasibility Studies; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids; D011107:Polylysine; D014162:Transfection / Q000379:methods*
chemical_list: D018952:Antigens, CD34; D019496:Cancer Vaccines; D002411:Cation Exchange Resins; D002412:Cations; D008055:Lipids; C086724:Lipofectamine; D011107:Polylysine; D000469:Alkaline Phosphatase