Towards RNA delivery via bacterial secretion systems
File(s)
Author(s)
Walker, Barnabas James
Type
Thesis
Abstract
RNA is a versatile macromolecule with numerous therapeutic applications. However, delivering RNA and RNA:protein complexes efficiently into the appropriate cells and cellular compartments remains a significant challenge. As our ability to engineer bacteria has become more sophisticated, there has been increasing interest in utilising bacteria as intelligent therapeutic agents in order to capitalise on many advantageous characteristics acquired over the course of millions of years of evolution. In particular, several bacterial secretion systems have evolved the ability to deliver complex macromolecules such as proteins and DNA across cell membranes and into target cells.
In this thesis, we explore ways of expanding these delivery capabilities further by investigating ways in which bacterial secretion systems could be harnessed for delivery of RNA and RNA:protein complexes. The type IV secretion system is able to deliver DNA into bacterial and eukaryotic cells via a covalent linkage to a secreted protein. In an attempt to mimic this process, we explore the use of a previously characterised synthetic ribozyme to ligate RNA to secreted proteins. Whilst we were able to confirm functioning of the ribozyme in vitro, no evidence could be found for ligation occurring in vivo. In a second approach, we work with the type VI secretion system (T6SS) which is unique in its ability to deliver proteins in a folded state and may therefore be able to deliver ribonucleoprotein complexes such as Cas9 with a guide RNA. A new assay for studying the translocation of heterologous proteins into bacteria via the T6SS is developed and used to test several T6SS substrates for their ability to support the delivery of Cre recombinase. None are able to do so, however a new cloning strategy is developed to facilitate higher throughput testing of candidate carrier proteins. Delivery of heterologous proteins into human cells is also explored via a previously published assay. We find that the readout of the assay is obscured by the intrinsic fluorescence of the bacteria used and make recommendations for a new assay.
In this thesis, we explore ways of expanding these delivery capabilities further by investigating ways in which bacterial secretion systems could be harnessed for delivery of RNA and RNA:protein complexes. The type IV secretion system is able to deliver DNA into bacterial and eukaryotic cells via a covalent linkage to a secreted protein. In an attempt to mimic this process, we explore the use of a previously characterised synthetic ribozyme to ligate RNA to secreted proteins. Whilst we were able to confirm functioning of the ribozyme in vitro, no evidence could be found for ligation occurring in vivo. In a second approach, we work with the type VI secretion system (T6SS) which is unique in its ability to deliver proteins in a folded state and may therefore be able to deliver ribonucleoprotein complexes such as Cas9 with a guide RNA. A new assay for studying the translocation of heterologous proteins into bacteria via the T6SS is developed and used to test several T6SS substrates for their ability to support the delivery of Cre recombinase. None are able to do so, however a new cloning strategy is developed to facilitate higher throughput testing of candidate carrier proteins. Delivery of heterologous proteins into human cells is also explored via a previously published assay. We find that the readout of the assay is obscured by the intrinsic fluorescence of the bacteria used and make recommendations for a new assay.
Version
Open Access
Date Issued
2020-10
Date Awarded
2021-06
Copyright Statement
Creative Commons Attribution 4.0 International Licence
License URL
Advisor
Polizzi, Karen
Stan, Guy-Bart
Freemont, Paul
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)