Use of cell membrane permeating peptides for delivery of plasmid DNA into dendritic cells

Immune therapy is a promising approach in the treatment of cancer. Besides ex vivo incubation of dendritic cells (DC) with tumor-associated antigens (TAA) and adjuvants, an increasing number of nanosized systems that... [ view full abstract ]

Immune therapy is a promising approach in the treatment of cancer. Besides ex vivo incubation of dendritic cells (DC) with tumor-associated antigens (TAA) and adjuvants, an increasing number of nanosized systems that co-deliver these compounds are assessed for effective in vivo drug delivery. DNA vaccines that encode for TAAs are an inexpensive, stable and safe alternative to protein- and peptide-based vaccines. Still, efficient delivery of pDNA in vivo is a challenge that needs to be faced.
LAH4-L1 is a cell membrane permeating peptide shown to complex pDNA and RNA. We analysed the suitability of this peptide to deliver pDNA to DC. First, we incubated murine spleen cells with fluorescently labelled LAH4-L1/pDNA complexes and analysed transfer of fluorescently labelled pDNA to different immune cell subtypes via flow cytometry. Due to strong interaction with endocytically active macrophages and DC, we also analysed bone marrow-derived dendritic cells (BMDC). BMDC showed strong binding/internalization of LAH4-L1 complexed pDNA. Unexpectedly, these BMDC also showed strong upregulation of surface markers that indicate cellular activation (MHCII, CD40, CD80, and CD86). In agreement, LAH4-L1/pDNA-treated BMDC generated strongly enhanced levels of proinflammatory cytokines (IL-1ß, TNF-a, IL-6). These findings suggest that LAH4-L1 may exert a potent stimulatory capacity on DC which is crucial for the induction of an adaptive immune response. LAH4-L1 mediated stronger transfection efficiency than JetPEI, often used for the transfection of DC-like cell lines with transgene-encoding pDNA, as assessed using a luciferase reporter. Ongoing work is focussed on the use of LAH4-L1 for transfection of DC populations with antigen-encoding mRNA and pDNA and subsequent DC/T cell co-cultures to assess their T cell stimulatory capacity.
Future work is focussed on functionalization of LAH4-L1 with targeting moieties to target DC specifically. Furthermore, TAA-encoding pDNA will be used for in vivo proliferation assays and to induce tumor-specific immune responses.