Rethinking Gene Expression Using the Synthetic C3P3 Transcription System

Jais_PhilippeDr. Philippe Jais, President and CSO of Eukarÿs SAS, recently spoke with Cambridge Healthtech Institute on the early challenges and successes in systems biology, the benefits of Eukarÿs’ C3P3 method, and the future of synthetic biology in both therapeutic and non-therapeutic applications. Dr. Jais will be speaking during the Cell Line and Systems Engineering conference at PEGS Europe, 18-19 November 2019.

1. What were some of the early challenges in eukaryotic artificial expression systems?

C3P3 (Chimeric Capping Prone Phage Polymerase) is the first and only non-viral artificial eukaryotic expression system ever developed. This artificial system generated by synthetic biology allows the autonomous production at high rate of messenger RNA (mRNA), and therefore protein(s) of interest. Unlike prokaryotic RNA, eukaryotic mRNA undergoes multiple post-transcriptional modifications, which account for its extreme complexity. These various modifications are essential to the biological activity of the mRNA and more particularly to its recognition by the translational machinery of the cell. In other words, mRNA without any post-transcriptional modification results in the expression of any protein. The main challenge to the development of the C3P3 system was to generate these various modifications of the mRNA. Because of the complexity of these mutations, C3P3 system was developed by successive generations. Each generation performs an additional type of modification and thus increases system performance. Schematically, the capping was optimized during the first generation of the system (C3P3-G1), which was a real challenge because of the complexity of this modification, polyadenylation by C3P3-G2, cell signaling by C3P3-G3 and epitranscriptomics by the current C3P3-G4.

2. How has synthetic biology enabled the development of Eukarÿs’ expression system?

The development of the C3P3 system was virtually impossible until the emergence of systemic biology in the late 2000s. The C3P3 system has two components: an artificial enzyme and DNA with the gene(s) of interest under control of the C3P3 enzyme. Both the enzyme and the DNA consist of multiple blocks that have to be individually optimized one after the other. The synthesis of these blocks was carried out by synthetic biology. For example, the first generation of the C3P3 system consisted of about twenty blocks produced by synthetic biology, the optimization of each block requiring about 3 months, and so more than five years for this generation alone. Great patience was therefore essential for the development of the C3P3 system!

3. What are the benefits of using an in vivo method like C3P3?

One main advantage of the C3P3 system lies in its performance. Depending on the cell line tested, the current expression levels range from 3 to 10 times higher than standard transient expression plasmids with CpG-rich matrices used for bioproduction and even greater with CpG-free DNA templates used for synthetic gene therapy, respectively. The C3P3 system also allows the production of non-coding RNAs which can be used for the purpose of genetic inhibition (shRNA, miRNA, lncRNA, trans-cleaving ribozymes, trans-splicing ribozymes, aptamers) and possibly also appropriate for genome engineering by the CRISPR/cas9 editing system. We make extensive use of these features in synthetic gene therapy. Lastly, the C3P3 enzyme also acts as an orchestra conductor, which makes possible the simultaneous expression and/or repression of several genes. It is therefore possible to inhibit gene expression, as well as to recombine mRNA or DNA with the C3P3 system. This ability to act at different levels makes the treatment of human multifactorial disorders by synthetic gene therapy possible.

4. What are the future applications of C3P3 and synthetic biology in general?

The C3P3 instrument currently available is well suited to the production of recombinant viruses, as well as multi-protein complexes such as VLPs (virus like particles) for vaccination. The next instruments to be developed will be more specifically dedicated to the bioproduction of recombinant proteins and, above all, monoclonal antibodies. The C3P3 system is also used for a new therapeutic approach called synthetic gene therapy. It relies on the use of synthetic DNA templates, which contains both the C3P3 gene and the gene(s) of interest under control of the C3P3 promoter. The modular structure of these treatments makes them usable with minimal adaptation for the majority of human diseases. Synthetic gene therapy is a radically innovative therapeutic approach that is designed as safe, non-viral, non-integrative, well-tolerated, self-vectorized, and produced at an acceptable cost. Although synthetic gene therapy can be used for the treatment of the majority of human diseases for which susceptibility genes have been identified, Eukarÿs is developing a pipeline initially dedicated to the treatment of certain liver diseases and cancers. Our most advanced product, EUK-LPR, demonstrated excellent performance in animals and soon will begin its regulatory preclinical phase. The launch of R&D phases for two new products for liver disease and cancer will be announced soon. The C3P3 system can be used in a very large number of other therapeutic or non-therapeutic applications. Due to the structural conservation of mRNA and its modifications throughout the eukaryotic kingdom, the C3P3 system is potentially usable after adjustments in all eukaryotic species, which includes yeasts, protozoa, plants, and animals. Its use can therefore be considered for human or animal vaccination, bioproduction in various eukaryotic species, in vitro protein synthesis, cellular functional tests, etc.

Philippe Jais, MD, PhD, is an Hepato-Gastroenterologist (Paris VII) and human Molecular Geneticist (INSERM, Gustave Roussy Institute, Johns Hopkins). He held various research management positions in the biotechnology industry (Genset, ProteaBio) and Pharma (Solvay, Abbott, Roche). He is the founder of Eukarÿs and the inventor the various generations of the C3P3 system, together with others’ technologies used for synthetic gene therapy with the C3P3 system.