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Cell therapies are taking center stage in medicine spanning cancer immunotherapy, stem cell engineering, and regenerative medicine. Recently, the first ex vivo hematopoietic stem cell (HSC) gene therapy for the treatment of adenosine deaminase deficiency-severe combined immunodeficiency (ADA-SCID) received marketing approval.1 By now, the majority of the 150 plus patients who have received HSC gene therapy to treat monogenic diseases have demonstrated clinical benefits.2 Promising clinical outcomes in phase I/II trials based on T cells engineered to express T cell chimeric antigen receptors (CARs)3 against hematological malignancies have also spurred unprecedented interest from pharmaceutical and biotechnology companies.

Controlled modulation of T-cell response during immunotherapy, especially the balance between T helper 1 (Th1) and Th2 responses, is critical for generating effective immune response. Here we report that dual delivery of interleukin 10 (IL-10)-targeted small interfering RNA (siRNA) and DNA vaccines to dendritic cells (DCs), using a single particle carrier, efficiently enhances immune response and modulates it toward a stronger Th1 phenotype. Surface-functionalized polymer microparticles (MPs) carrying both IL-10-targeted siRNA and DNA antigens exhibited effective gene silencing, DNA transfection, and synergistically enhanced upregulation of maturation markers in primary DCs leading to increased T-cell proliferation, in vitro. Mice immunized with these dual-delivery carriers demonstrated a significant "switch" toward Th1 response as evidenced by increase in interferon gamma (IFN-gamma) production and decrease in IL-4 production by CD4+ T cells. This further led to enhanced antiviral cytotoxic T-lymphocyte activity. Such dual siRNA-DNA delivery provides a novel strategy to precisely control the type and strength of T-cell response during immunotherapy.

Two polyethylene oxide-based delivery systems comprised of reacting PEG polymers were designed for the delivery of DNA expression vectors. The polymers are formulated with the DNA and injected into the muscle, wherein a crosslinked matrix forms in-situ. The matrix resembles a viscous solution (formulation A) or a gel (formulation B). The reacting PEG polymers do not interact with, but entrap the DNA. The formation of the matrix does not affect the supercoiling of the incorporated DNA. The polymers are biocompatible and biodegradable due to the presence of hydrolytically labile bonds in one of the components. Measurement of degradation in vivo suggests that a significant amount of the polymer disappears from the injected muscle by 28 days post injection. Administration to mice of SEAP plasmid DNA formulated with the PEG polymers results in SEAP expression. Expression levels are similar to those of unformulated DNA, but the duration of gene expression is significantly longer in immunocompetent animals receiving the formulated DNA. Significantly lower anti-SEAP IgG titers are elicited by network-formulated DNA relative to unformulated DNA, even though expression levels are comparable. The data suggests that the matrix extends duration of expression by reducing the anti-SEAP immune response so that these delivery systems may be useful for prolonged gene expression following a single intramuscular injection.

Top of pageAbstract Introduction: We earlier demonstrated that branched Polyethyleneimine (PEI) conjugated poly(lactide-co-glycolide)PLGA microparticles enhanced transfection in phagocytic RAW cells in vitro.1 We present herecomparison of the effects of linear and branched PEI conjugated PLGA microparticles in enhancing the potential of a model idiotype/chemokine fusion encoding genetic vaccine for B cell lymphoma in mice models. Branched PEI70kDa polymer conjugated PLGA microparticles in comparison with linear PEI conjugated microparticles; a) better adsorbed plasmid DNA b) showed significant adjuvant effects by upregulating MHC class II and CD80 molecules in macrophage cells and finally c) demonstrated enhanced anti tumor effects upon challenge with an A20 B cell lymphoma tumor in Balb/C mice in vivo. Branched PEI70kDa conjugated PLGA microparticles imparted anti tumor effects which were comparable or slightly better than gene gun mediated immunization, especially when administered intramuscularly. Materials and Methods: Synthesis of cationic microparticles and characterization was performed as described earlier1. FACS studies were conducted using fluorescent antibodies such as MHC class II, CD80, F4/80 (Ebiosciences, CA) were used to stain RAW264.7 murine macrophage cells (ATCC, VA) post formulation stimulation. Balb/c mice 8-10 weeks old were injected 3 times with cationic microparticles and with gene gun based approaches. A20 tumors were injected at 2.5 times the minimal lethal dose. Survival rates of mice (anti tumor effects) were followed and plotted to confirm statistical significance. Results and Discussion: Plasmid DNA adsorbed on 70kDa branched PEI modified PLGA microparticles demonstrated significant upregulation of MHC class II and CD80 molecules in RAW macrophage cell lines in vitro. These results were also consistent with potent anti tumor effects in vivo. Intramuscular injections of cationic microparticles with adsorbed vaccine encoding plasmid DNA demonstrated anti tumor effects which were comparable with long established gene gun based approaches. Intramuscular injections of cationic particle based formulation proved to be superior to intradermal injections. Conclusions: Cationic PEI conjugated PLGA microparticles could offer an attractive strategy to enhance the immunogenecity of weak self antigens encoded by cancer DNA vaccines. We hypothesize that these alternative approaches with injectable formulations offer more flexibility in terms of introducing additional adjuvants such as synthetic MPL-A and CpG optimized oligos which could significantly further enhance the potential of these cancer genetic vaccines. Current studies are underway to test these enhanced anti tumor effects by co-administration of soluble adjuvants.

Introduction: Biodegradable poly (lactide-co-glycolide) PLGA based particulate delivery systems have been observed to be promising adjuvants when used in peptide/protein or plasmid DNA based approaches in genetic immunization1. We have designed and evaluated a rational approach for surface modification of PLGA microparticles with covalent immobilization of branched and linear PEI which a) imparts a cationic charge to the microparticles b) improves buffering ability compared to unmodified PLGA microparticles c) mediates possible endosomal escape as evaluated by confocal fluorescent microscopy d) imparts minimal cyotoxicity and e) mediates improved exogenous gene transfer in murine macrophage cell lines.