Alphavirus (i.e., Venezuelan encephalitis virus, Sindbis virus, Semliki Forest virus, etc.) self-replicating RNA replicons (also called Self-Amplifying mRNA) are promising technology platforms for vaccine development. A multitude of pre-clinical studies have highlighted the potency of replicon vaccines in numerous models of infectious diseases and cancer1. Many RNA replicon vaccines are currently entering human clinical trials. Self-replicating (self-amplifying) RNA replicons are genetically engineered viral RNA molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions (infectious virus particles).
For instance, cell lines harboring replicating hepatitis C virus (HCV) RNA replicons were used to produce direct-acting antivirals capable of controlling viral infection at least short-term. As both HCV and alphaviruses belong to positive-strand RNA viruses – their replication strategies are extremely similar. For HCV, the viral RNAs entering the cytosol of infected cells were rapidly degraded. In particular, an initial 10-fold decrease in viral RNA genomes post-infection was observed2, whereas replicons delivered using electroporation were decreased by up to ~100-fold3. Subsequently, the components of viral replication machinery (the replicase) encoded by either viral RNA genomes or replicons have to be efficiently translated by the host cell ribosomes. Then, the components of viral replication machinery assemble at the intracellular membranes of host cells, recruit viral template or replicon RNA, and induce multiple invaginations harboring replication complexes (also called viral factories), which are connected to the cytosol4. These numerous virus-induced vesicles are termed “spherules”5 and “double-membrane vesicles”6, for alphavirus and HCV, respectively. Inside the vesicles, viral replication machinery functions to generate multiple full-length copies of the viral genome or replicon RNA. For HCV, the abundant induction of vesicles by its viral replication machinery does not depend on RNA replication (no viral or replicon RNA template must be present)6, whereas abundant alphavirus spherules are produced only when viral or replicon RNA is actively replicated7. It was demonstrated that alphavirus replication factories can generate ~150,000 copies of genomic and subgenomic (encoding the gene of interest, i.e., the antigen) RNAs per single cell in just under 8 hours after infection8.
Several important conclusions can be drawn based on the facts presented above. First, the degradation of replicon RNA molecules in the cytosol of host cell is a natural process essential for establishing robust replication. Second, replicon RNA molecules must be delivered directly to the cytosol to enable the efficient translation of replication machinery components. Third, the recruitment of the replicon RNA as well as the assembly of the replication factories requires an intricate series of events that involve viral replication machinery, replicon RNA, and host membrane lipids interactions.
In this Solution, NextGenRnD proposes novel RNA vaccine formulation that: (i) is based on FDA compliant materials; (ii) should be compatible with encapsulation of RNA molecules ranging in size from 700 nucleotides (nts) to approximately 24,000 nts; (iii) can be lyophilized and resuspended when necessary; (iv) can be used for subcutaneous or intramuscular delivery; (v) is low cost, i.e., the cost of materials for 0.5 milligram (mg) RNA encapsulation is approximately $1.