Cell free DNA manufacturing to control sequence and topology and improve transfection efficiency / product quality in cell and gene therapy applications

Project ID: 2228cc1447 (You will need this ID for your application)

Under Offer

Research Theme: Manufacturing The Future

UCL Lead department: Biochemical Engineering

Department Website

Lead Supervisor: Daniel Bracewell

Industry partner: FUJIFILM Diosynth Biotechnologies

Stipend enhancement: £3,500 pa

Project Summary:

Cell-free synthesis (CFS) uses enzymes to generate biological products, including proteins, mRNA and plasmid DNA (pDNA). This contrasts with the existing paradigm where cell factories (microbial or mammalian) are used to synthesize such products. For plasmid DNA the approach allows replication and amplification from a template sequence, without the restrictions imposed by cells particularly antibiotic resistance sequences that are of regulatory concern. Additionally, CFS has a number of features that enable distributed, small-scale manufacture critical to the trajectory of the pharma industry towards personalised medicines: 1) In contrast to cell-based manufacture, which is time consuming, complex, variable and requires bulky equipment, cell-free synthesis is fast, simple to set up, and reproducible.
2) Cell-Free pDNA can also be produced to a higher initial purity level, simplifying downstream processing and the number of process related impurities. 3) Cell and Gene Therapies are reliant on pDNA for viral vector manufacture, avoiding E.coli based manufacture simplifies the complicated supply chain. Using a novel cell free DNA synthesis technology developed in Bracewell’s group in the Department of Biochemical Engineering at UCL it is possible to produce various forms of pDNA using a novel reactor design. This enables the exploration of how this impacts transfection in different systems using the experience of those in Waddington’s Gene Transfer Group at UCL’s Institute of Women’s Health and at Fujifilm Diosynth. The research will build on published observations from Waddington’s group which shows a considerable difference in expression in mice models between cell-free DNA and plasmid DNA produced using E.coli to elucidate the mechanisms underlying this difference. Using the adeno-associated virus (AAV) system with Fujifilm Diosynth differences in DNA topology (supercoiling), methylation, size and sequence will be investigated. For example, via use of gyrase enzymes to rationally manipulate the secondary structure of the DNA.