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Development of broad-spectrum antimicrobials using modified antisense oligonucleotides

Development of broad-spectrum antimicrobials using modified antisense oligonucleotides
Development of broad-spectrum antimicrobials using modified antisense oligonucleotides
Antibiotics have formed a corner stone of modern medicine. However, bacteria can develop resistance against antibacterial drugs and new antibiotics have to be created at all times to compete with this resistance. One way of potentially generating new antibiotics is by using antisense oligonucleotides (ASOs) that can recruit enzymes to cleave the mRNA of essential genes. In chapter 2, we thus designed and synthesized a number of PNA-based ASOs targeting essential, reporter and virulence genes of E. coli. The cell penetrating peptide KFFKFFKFFK was covalently attached to the PNAs in order to facilitate the uptake of the oligonucleotides into the bacterial cells. Using a variety of antibacterial assays, we were able to show that PNAs functioning by sterically blocking the ribosome binding site are able to silence the activity of the ftsZ and katG gene. Unfortunately, PNAs that function by recruiting RNase P via the external guide sequence did not seem to have any gene silencing ability. In vitro studies suggested that this is because the PNA backbone cannot be recognized by the RNase P ribozyme. In chapter 3 we therefore tried to recruit a different enzyme, RNase H. A number of LNA-DNA-LNA gapmers targeting the ftsZ gene in E. coli were designed and synthesized. The standard RNase H recruitment assay showed that these oligonucleotides are able to induce cleavage of the target mRNA by the enzyme. Microscopy studies confirmed that the gapmers are also able to induce gene silencing in bacteria, presumably through the recruitment of RNase H. In Chapter 4, we systemically designed and screened libraries of chemically modified oligonucleotides containing the external guide sequence for their ability to recruit RNase P in vitro. We had a number of successful hits, mainly hybrids of DNA, LNA and to a lesser extent OMe. Due to the high DNA content of the hits, the potential dual recruiting of RNase P and RNase H was explored. A number of chemically modified oligonucleotides were identified that can induce in vitro cleavage of their target mRNA by both enzymes. Such compounds are expected to have superior gene silencing ability. Microscopy studies confirmed the in vitro findings, as we were able to detect elongated E. coli cells (indicative of successful ftsZ gene silencing) for those compounds that showed potential dual recruitment activity. The work presented in this thesis has shown a promising technology for the use of chemically modified antisense oligonucleotides as antibiotics, especially when their gene silencing could be enhanced by the recruitment of RNase P, RNase H or both. However, the biggest challenge for the development of any oligonucleotide based therapeutic remains their problematic delivery into the target cells or bacteria. Future efforts will thus have to focus on identifying more efficient ways of delivering oligonucleotides into bacterial cells.
University of Southampton
Gneid, Hassan
1f4ee688-0336-495e-b112-3309387d8076
Gneid, Hassan
1f4ee688-0336-495e-b112-3309387d8076
Watts, Jonathan K
c4de85ee-aaa3-4e7d-99b3-147a4de4f01c

Gneid, Hassan (2019) Development of broad-spectrum antimicrobials using modified antisense oligonucleotides. Doctoral Thesis, 280pp.

Record type: Thesis (Doctoral)

Abstract

Antibiotics have formed a corner stone of modern medicine. However, bacteria can develop resistance against antibacterial drugs and new antibiotics have to be created at all times to compete with this resistance. One way of potentially generating new antibiotics is by using antisense oligonucleotides (ASOs) that can recruit enzymes to cleave the mRNA of essential genes. In chapter 2, we thus designed and synthesized a number of PNA-based ASOs targeting essential, reporter and virulence genes of E. coli. The cell penetrating peptide KFFKFFKFFK was covalently attached to the PNAs in order to facilitate the uptake of the oligonucleotides into the bacterial cells. Using a variety of antibacterial assays, we were able to show that PNAs functioning by sterically blocking the ribosome binding site are able to silence the activity of the ftsZ and katG gene. Unfortunately, PNAs that function by recruiting RNase P via the external guide sequence did not seem to have any gene silencing ability. In vitro studies suggested that this is because the PNA backbone cannot be recognized by the RNase P ribozyme. In chapter 3 we therefore tried to recruit a different enzyme, RNase H. A number of LNA-DNA-LNA gapmers targeting the ftsZ gene in E. coli were designed and synthesized. The standard RNase H recruitment assay showed that these oligonucleotides are able to induce cleavage of the target mRNA by the enzyme. Microscopy studies confirmed that the gapmers are also able to induce gene silencing in bacteria, presumably through the recruitment of RNase H. In Chapter 4, we systemically designed and screened libraries of chemically modified oligonucleotides containing the external guide sequence for their ability to recruit RNase P in vitro. We had a number of successful hits, mainly hybrids of DNA, LNA and to a lesser extent OMe. Due to the high DNA content of the hits, the potential dual recruiting of RNase P and RNase H was explored. A number of chemically modified oligonucleotides were identified that can induce in vitro cleavage of their target mRNA by both enzymes. Such compounds are expected to have superior gene silencing ability. Microscopy studies confirmed the in vitro findings, as we were able to detect elongated E. coli cells (indicative of successful ftsZ gene silencing) for those compounds that showed potential dual recruitment activity. The work presented in this thesis has shown a promising technology for the use of chemically modified antisense oligonucleotides as antibiotics, especially when their gene silencing could be enhanced by the recruitment of RNase P, RNase H or both. However, the biggest challenge for the development of any oligonucleotide based therapeutic remains their problematic delivery into the target cells or bacteria. Future efforts will thus have to focus on identifying more efficient ways of delivering oligonucleotides into bacterial cells.

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HassanGneid-Final PhDThesis
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Published date: September 2019

Identifiers

Local EPrints ID: 447168
URI: http://eprints.soton.ac.uk/id/eprint/447168
PURE UUID: 4f8297e9-9ae3-4c58-b2a2-e4d66e8c902f

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Date deposited: 04 Mar 2021 17:39
Last modified: 04 Mar 2021 17:39

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Contributors

Author: Hassan Gneid
Thesis advisor: Jonathan K Watts

University divisions

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