Biology:Chimeric cytoplasmic capping-prone phage polymerase expression system
The chimeric cytoplasmic capping-prone phage polymerase (also named C3P3 system) is an artificial eukaryotic expression system, which synthesizes mature messenger RNA (mRNA) of interest in the cytoplasm of eukaryotic host-cells.[1][2] mRNA thus synthesized conveys the genetic information to the ribosome, where it specifies the amino acid sequence of the protein products of gene expression.
The C3P3 expression system can be used for living eukaryotic cells (in cellulo) or organisms (in vivo), but so far its uses has been only reported for mammals. However, due to conservation of mRNA structure and its modifications in the eukaryotic kingdom that mimed by this system, its use has been proposed for other species eukaryotic species (e.g. yeasts, plants or other animals).[3] The C3P3 system consists of two components: firstly, an artificial enzyme produced by synthetic engineering and secondly specific DNA templates that contain a coding sequence under control of the C3P3 promoter . The specific DNA templates are therefore transcribed by the C3P3 enzyme and generate the mRNA of interest in the cytoplasm of host-cells. When assembled these two components synthesize high levels of mature messenger RNA and/or non-coding RNA in the host-cell cytoplasm without using the host cell transcriptional machinery.
In its first generation, the C3P3 enzyme would be a single-subunit protein made by the fusion of the African swine fever virus (AFSV) capping enzyme with a mutant DNA-dependent RNA polymerase from the K1E bacteriophage.[4][5] Such C3P3 enzyme produces mRNA capping modifications, which are found at the 5’-ends of mRNA and are strictly required for the translation of eukaryotic mRNA, i.e. the production of proteins. The second generation of the C3P3 system would allow polyadenylation at 3’-ends, which is also important for translation. A third generation has been announced, but no details have been made public.[6][7]
The C3P3 system has been used for bioproduction of recombinant proteins and reverse genetics of RNA viruses including Orthoreovirus [8] and rotavirus.[9] This system is also tested in animals in order to treat or prevent human monogenic and multifactorial diseases by synthetic gene therapy.[10]
References
- ↑ Jaïs, Philippe H.; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila et al. (2019-02-06). "C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system" (in en-US). Nucleic Acids Research (US) 47 (5): 2681–2698. doi:10.1093/nar/gkz069. PMID 30726994.
- ↑ Elroy-Stein, Orna; Moss, Bernard (1990). "Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells" (in en). Proceedings of the National Academy of Sciences 87 (17): 6743–6747. doi:10.1073/pnas.87.17.6743. PMID 2204064. Bibcode: 1990PNAS...87.6743E.
- ↑ https://biosynsys2016.sciencesconf.org/111450/document
- ↑ Jaïs, Philippe H.; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila et al. (2019-02-06). "C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system" (in en-US). Nucleic Acids Research (US) 47 (5): 2681–2698. doi:10.1093/nar/gkz069. PMID 30726994.
- ↑ https://biosynsys2016.sciencesconf.org/111450/document
- ↑ Artificial C3P3 transcriptional engine for synthetic gene therapy. Global Synthetic Biology & Gene Editing Congress 2017, London, United Kingdom.
- ↑ http://eukarys.com/wp-content/uploads/2019/02/C3P3-G3_Press_Release.pdf
- ↑ https://jvi.asm.org/content/91/11/e02416-16
- ↑ https://jvi.asm.org/content/early/2020/07/10/JVI.01294-20?versioned=true
- ↑ Synthetic gene therapy: a novel approach for the treatment of human liver disorders. American Society of Gene & Cell Therapy 2016, Washington, DC.
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