Exploring Vibrio natriegens potential as a new microbial chassis for plasmid DNA production

Exploring Vibrio natriegens potential as a new microbial chassis for plasmid DNA production Simão Pedro Fonseca1*, Ana Rita Silva-Santos1, Ricardo Silva1, Gabriel Monteiro1 and Duarte Miguel Prazeres1 1 iBB – Insitute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisbon, Portugal *[email protected] RESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODS MOTIVATION BACKGROUND Plasmid DNA has long played a key for the biotech industry and for scientific research. While intially used as a cloning vector, it is currently fundamental for novel, state-of-the-art therapies as most of these rely, at some point, in plasmid DNA: Manufacturing Process Final Application Large-scale manufacture of plasmid DNA has become the bottleneck for the biopharmaceutical industry. This process is currently done using E. coli as a host, exclusively. The growing demand of pDNA encourages the search and development of a novel, more suitable, host: 1. TRANSFORMATION 2. EXPANSION 4. TRANSFECTION Control E. coli V. n at r i e g en s Recombinant Proteins mRNA vacines Fastest-growing organism knwon to date Metabolically proficient Non-pathogenic Vibrio natriegens EU ED VD VU Acknowledgements: Research Unit Institute for Bioengineering and Biosciences – iBB acknowledges funding from FCT in the scope of projects UIDB/04565/2020 and UIDP/04565/2020 and LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. This project has received funding from the European Union's Horizon Europe, grant number 101159993. V. n a tr ie g en s cell were successfully transformed with pVAXGFP, encoding for a reporter GFP designed for mammalian transfection. pVAXGFP replicated in V. n a tr ie g en s showed a similar topology to that replicated in E. coli. Supercoiled isoforms of pVAXGFP replicated in V. n at r ie g en s were able to drive GFP expression in HEK293T cells. Fluorescence intensity was comparable to that obtained with plasmid replicated in E. coli, indicating a similar level of transgene expression. pVAXGFP V. n a t r i e g e n s Transformation Expansion Alkalyne Lysis Primary Purification Chromatography Transfection HEK293T Fluorescent Microscopy Cell & Gene Theraphy Immunotheraphy 3. PURIFICATION V. n a tr i eg e ns cells produced reasonable amounts of pDNA, although still lower quantity than that of E. coli. The percentage of supercoiled isoforms recovered from total pDNA for both bacteria was similar (≈25%). E – E. coli; V – V. natriegens; U – Undigested; D – Digested. EF E3 E14 E24 VF V3 V14 V24 Supercoiled isoforms of pVAXGFP were purified by multimodal chromatography from a feed stream containing multiple pDNA isoforms and RNA, for both bacteria. Fractions recovered had >96% sc pDNA and were virtually gDNA and protein free. V. n a tr iege n s was transformed with a plasmid designed for mammalian transfection and showed a similar topology to that replicated in E. coli. Purified supercoiled isoforms of the plasmid obtained from V. natriegens were complaint with industry standards and able to induce comparable levels of transgene expression in HEK293T cells. Although productivity was lower for V. na t ri g en s , this bacterium can definetely be used as a host for the manufacturing of plasmid DNA and is a promising alternative to E. coli. F – Feed; # - Corresponding Fraction. RNA sc oc