Novel immmunization strategy, using non-viral carriers for mRNA, encoding HIV protein variants.

We have developed an experimental immunotherapy for HIV, based on the ex vivo electroporation of patientmonocyte-derived dendritic cells (Mo-DC) with mRNA encoding consensus orautologous HIV gag mRNA. Presently, macaque and human phase I/II clinicaltrials are ongoing. This strategy is very promising because it is safe, it cancover the quasispecies and new immune responses against the virus can beinduced. The latter two elements are important to avoid immune escape. The drawback, however, is that ex vivo loading of DC with mRNA involves ahighly complex methodology and in vivo electroporation of DC is notfeasible either. Therefore, we now propose to transduce DC in vivo with mRNA, using non-viral carriers. Such carriers, mainly based on cationic polymers and cationic lipids, should bind tightly to mRNA thereby forming nanosized particles, so called lipoplexes and polyplexes.  These kinds of nanoparticles have already successfully been used to transfect cells in vivo with plasmid DNA and siRNA, showing that they can indeed deliver intact nucleic acids to cells. However, this result cannot immediately be extrapolated to mRNA, since the physicochemical properties of mRNA, siRNA and plasmid DNA molecules do significantly differ from each other, e.g. siRNA is composed of only 20-25 nucleotides while mRNA contains1000 of nucleotides. On the other hand siRNA has to reach the cytosol, plasmid DNA has to be delivered in the nuclei of cells, whereas mRNA has to arrive into the cytoplasm. A first challenge of this project is the design of non-viral carriers that can protect mRNA from extra- and intracellular degradation and deliver it to the ribosomes in the cytoplasm.

The non-viral mRNA carriers will be carefully characterized in terms of size, potential and stability. In addition, their efficiency to mediate effective mRNA cell transfer and protein expression will be evaluated by flowcytometry. Further on, also the mechanism by which mRNA carrying complexes internalize will be investigated. For plasmid DNA carrying complexes it was shown that they could be internalized by respectively clathrin- and caveolae endocytosis, but only the latter endocytosis route resulted in efficient transfection. To define pathways involved in uptake of mRNA complexes,specific inhibitors will be used to block the possible internalization pathways and colocalization studies will be performed with confocal fluorescencemicroscopy. Next, the immunogenicity of the mRNA particles will be investigated in cell cultures from both healthy donors and HIV seropositive donors. After culturing Mo-DC with HIV gag mRNA carrying lipo-orpolyplexes, the DC will be cocultured with autologous T cells. The induction of primary (healthy donors) and recall (seropositive donors) T cell responses towards HIV Gag peptides will be verified by ELISPOT. Furthermore also the quality of the immune response will be investigated by measuring polyfunctionality, virus inhibition capacity and proliferation of T cells.

Critical parameters for efficient mRNA delivery towards DC are (i) the stability of the mRNA carriers at siteof injection, (ii) its uptake by local DC, and (iii) the migration ofDC to draining lymph node. Those 3 parameters will be investigated after injection of C57Bl/6 mice. In addition, the immunopotentiating outcome of this strategy will be evaluated in mouse models. Positive control mice,expressing a transgenic T cell receptor with specificity for ovalbumin-derived peptides (OT-I and OT-II strains) will be injected with ovalbumin mRNA carrying polyplexes. This allows for a highly sensitive detection of T cell activation in the organism. Later on, these studies will be extended to wild-type mice using subcutaneous, intramuscular and mucosal routes of administration of nonviral carriers complexed with HIV gag mRNA. If successful, immunogenicity studies in macaques will be carried out as part of a follow-up project.