Gene Therapy (2000) 7, 2105–2112  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt CELL-BASED THERAPY RESEARCH ARTICLE Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells KS Denis-Mize 1,2 , M Dupuis 1,2 , ML MacKichan 2 , M Singh 2 , B Doe 2 , D O’Hagan 2 , JB Ulmer 2 , JJ Donnelly 2 , DM McDonald 1 and G Ott 2 1 Department of Anatomy, and Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA; and 2 Vaccines Research, Chiron Corporation, Emeryville, CA, USA Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation con- sists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-gly- colide (PLG) and the cationic surfactant, cetyltrimethylam- monium bromide (CTAB). Using preparations of fluorescent- labeled plasmid DNA formulated on PLG-CTAB micropar- ticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of Keywords: DNA delivery; dendritic cells; cationic microparticles, DNA vaccines Introduction Dendritic cells (DC) have been characterized as the most potent antigen presenting cells of the immune system (reviewed in Refs 1 and 2). Specifically, DC efficiently internalize, process and present antigens, and also migrate to lymphoid organs, secrete cytokines, and express co-stimulatory molecules required for lympho- cyte signaling. Their central role in initiating potent immune responses by eliciting helper and cytotoxic T cells, antibodies, and IL-12, has made them logical targets for development of both prophylactic and therapeutic applications. 2 Targeting antigens into dendritic cells in vivo or exposing DC to antigen ex vivo may enhance the immunogenicity of vaccines or induce T cell-mediated anti-tumor responses for cancer immunotherapy. The development of methods to generate large num- bers of DC has allowed researchers to harness their potent immunostimulating properties. 3,4 The discovery that granulocyte–monocyte colony-stimulating factor (GM-CSF) was the key cytokine involved in differen- tiation of hematopoietic progenitors to dendritic cells Correspondence: G Ott, Chiron Corporation, 4560 Horton Street, M/S 4.3, Emeryville, CA 94608–2916, USA Received 12 March 2000; accepted 14 September 2000 the fluorescence was concentrated in endosomal compart- ments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was sub- sequently processed and presented, resulting in stimulation of an H-2k d -restricted, gag-specific T cell hybridoma. Acti- vation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1–20 g DNA (10– 2000 g PLG) and was sustained up to 5 days after trans- fection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells. Gene Therapy (2000) 7, 2105–2112. made it possible to generate dendritic cells from murine bone marrow (BMDC). 5,6 Since that time, many preclini- cal studies using mouse models have elaborated on the important role these cells play in generation of the immune response and demonstrated their potential use in antiıtumor immunity (reviewed in Ref. 7). Anti-tumor cytotoxic T lymphocyte activity and protection against lethal tumor challenge in mouse models have been dem- onstrated using cytokine-driven BMDC pulsed with tumor-associated peptides and whole tumor lysates transferred by the subcutaneous route. 8,9 Several clinical trials have established general safety and feasibility, and more are ongoing to demonstrate clinical efficacy of DC vaccination in several types of cancer including mela- noma, prostate, breast, lung, colorectal, and renal cell car- cinomas (reviewed in Ref. 7). Yet another strategy cur- rently in development involves the introduction of genes encoding antigens directly into DC, allowing direct priming of both cytotoxic and helper T cell responses. Although recombinant viral vectors or intracellular bacteria can efficiently deliver genes encoding antigen to DC and induce cell-mediated immunity, 10–12 issues of safety and manufacturability continue to be addressed. 13–16 In vitro transfection efficiency of DC by nonviral methods has been extremely poor. 17 While progress has been made by the use of electroporation, the efficiency of DC trans- fection is extremely low and results in substantial loss of