| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 106 === Among rapidly developed nucleic acid therapeutics, mRNA-mediated approach offers an attractive alternative to DNA-mediated gene therapy. The mRNA-based approaches have potential to increase translation efficiency and to lower insertional mutagenesis compared that to DNA-based therapies. However, the applications of mRNA are limited to its instability and the cost to produce. Therefore, a robust RNA delivery system is required for achieving RNA applications. An ideal RNA delivery system is expected to (i) protect cargo against ubiquitous nucleases, (ii) prevent un-wanted interaction with non-specific biomolecules, (iii) deliver cargo to tissues of interest, and (iv) promote cell entry efficiency.
Virus-like particles (VLPs) are macromolecular assemblies of non-infectious viral coat proteins (CP) in a highly ordered repetitive structural configuration. The VLPs derived from virus counterparts can be expressed in vivo and CPs are able to assemble into a complete particle spontaneously. Prior art VLPs are found being capable of packaging biological macromolecules such as proteins and nucleic acids. Here we use uricase (UOX) as a study case in this research. The UOX is an enzyme that catalyzes the oxidation reaction of uric acid and also regulates uric acid homeostasis in most mammalians. Here we propose to use VLPs to encapsulate the UOX messenger RNA (mRNA), deliver it to host cells via VLPs, and explore the feasibility of functional protein expression in eukaryotic systems.
In this study, the UOX and Qβ-CP genes are successfully designed and cloned into two plasmids (pT7CFE1-NHA-UOX and pCDF-1b-CP). These two constructs were co-transformed into E. coli strain BL21 to produce Qβ-VLPmUOX. We validated the assembly of the Qβ-VLPmUOX by using TEM technique. The cargo transcripts (mUOX_P and mUOX_SP) packed inside Qβ-VLPmUOX is verified by the denaturing PAGE. In addition, we showed that the cargo plasmid is capable of being transcribed and then translated into a functional UOX by utilizing the cell-free translation. The function of the in vitro expressed UOX is assayed positively via the AmplexRed uricase assay. Meanwhile, the Qβ-VLP-encapsulated mUOX shows no translational capability neither in cell-free translation system nor in cell culture despites the input mass of the cargo transcripts varies up to 2000 g. The goal of using Qβ-VLPmRNA for functional protein production in vivo remains a challenge and yet some obstacles discussed in the contents require to be further investigated and overcome in future work.
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