###Cell free DNA Synthesis to Understand and Improve Transfection Efficiency
Project ID: 2228bd1009 (You will need this ID for your application)
Research Theme: Manufacturing The Future
UCL Lead department: Biochemical Engineering
Lead Supervisor: Daniel Bracewell
Project Summary:
Cell-free synthesis (CFS) uses enzymes to generate biomolecules, including proteins, mRNA and plasmid DNA (pDNA), in vitro. The use of enzymes and primers allows plasmid DNA to be replicated and amplified considerably from a template sequence, without the need for any specific sequences to be present, i.e. it eliminates the issue of microbial sequences, particularly antibiotic resistance sequences. Additionally, CFS has a number of features that aid both in enabling distributed, small-scale manufacture and screening of candidate sequences: 1) In contrast to conventional cell-based manufacturing methods, which are time consuming, complex, variable and require bulky equipment, cell-free synthesis is fast, simple to set up, more reproducible and only requires basic equipment. 2) Cell-Free pDNA can also be produced to a higher initial purity level. 3) For C> applications, avoiding E.coli based manufacture in the same facility as mammalian culture and/or reducing the already complicated supply chain is an additional benefit to using CFS of DNA
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 investigate various production methods and forms of pDNA or RNA. This enables the exploration of how this can impact transfection in different systems using the experience of those in Waddington’s Gene Transfer Group.
Test case 1/ Building on published observations from S. Waddington’s group which shows a considerable difference in expression in mice models for cell-free DNA and traditional plasmid DNA produced using E.coli. The differences in 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.
Test case 2/ Quantify the degree of batch-to-batch variability between results from conventionally and CFS produced ASO RNAs for screening of constructs.