In Vitro-Reconstituted Virus-like-particle Delivery of Self-Amplifying RNA Genes
- Author(s): Biddlecome, Adam Min
- Advisor(s): Gelbart, William M
- et al.
The delivery of RNA genes has great potential in a range of therapeutic applications. A couple of main limitations for RNA-based therapies include its vulnerability to ribonuclease (RNase) digestion, and its transient and low expression levels due to its lack of amplification. Accordingly, a gene delivery platform that includes self-amplifying mRNA inside a protective capsid allowing for cell targeting and uptake could represent a major step forward in mRNA-based gene therapy.
The self-amplifying nature of viral-encoded RNA-dependent RNA polymerases (RdRp) of Nodamura virus holds promise in the development of a platform for self-amplifying RNA gene delivery. To protect and assist in uptake of mRNA encoding an RdRp coupled to a gene of interest, plant virus-like-particles (VLPs) are a strong candidate. Cowpea chlorotic mottle virus (CCMV) capsid protein has been shown to be able to package in vitro heterologous mRNAs, and also to make the genes available to mammalian cells. To better characterize the uptake and amplification in mammalian cells of RdRp-associated genes (also called a replicon, because of its self-amplifying nature), both when naked or encapsidated, reporter genes such as enhanced yellow fluorescent protein (EYFP) or luciferase were used.
In collaboration with the German pharmaceutical company Boehringer Ingelheim (B-I), evaluation of possible efficacy of this platform for a cancer vaccine was explored by incorporation of a model antigen (Ovalbumin epitope, SIINFEKL) sequence into the RNA1 of Nodamura virus. From in vitro studies involving incubation of OVA-replicon-VLPs, we observed that CCMV VLPs can be taken up by dendritic cells, where replication then occurs to promote increased production of antigen. Flow cytometry analysis revealed increased expression of maturation markers, and assaying the dendritic cell content by qPCR showed a significant number of SIINFEKL epitope-containing RNA. After these OVA-replicon-VLPs are injected into mice, a population of SIINFEKL-specific CD8+ (cytotoxic) T cells results.