sRNA-based therapeutics for disease caused by A. pleuropneumoniae

Lead Research Organisation: University of Portsmouth
Department Name: Inst of Biomedical and Biomolecular Sc

Abstract

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Technical Summary

Actinobacillus pleuropneumoniae (APP) causes acute and chronic lung disease in pigs and is of substantial economic importance to the worldwide swine industry. There is an urgent need for improved vaccines and therapeutics as antibiotic resistance is an increasing problem. The focus of this proposal is bacterial small non-coding RNAs (sRNAs) which, facilitated by Hfq, play a key role controlling mRNAs responsible for virulence. Our work has shown that the hfq-mutants of APP are severely attenuated and rapidly cleared from pigs. While Hfq could be targeted directly as a therapeutic approach, the presence of homologous eukaryotic proteins suggests that targeting the associated sRNAs will be a more effective strategy. In particularly, this approach will bring about a therapeutic effect without the risk of developing bacterial resistance, since no survival pressure, from which traditional antibiotic resistance originates, is imposed.

To date, our work has experimentally identified 15 Hfq-associated sRNAs in APP, and computationally a further 30 have been predicted. Our recent bioinformatic analysis confirms a number of these sRNAs target mRNAs with relevance to virulence. This proposal represents a major collaboration between Imperial College London and the University of Portsmouth, utilising the extensive experience in APP and Hfq-sRNA interactions, respectively. We plan to ascertain the identity of Hfq-associated sRNAs that are key in turning on virulence genes and determine whether inhibiting these sRNAs using peptide nucleic acids (PNAs) provides a novel therapeutic approach to treating APP disease. Using a combination of in vitro biochemical and biophysical molecular characterisation approaches, coupled with in vivo reporter and/or phenotypic assays, we will determine the viability of this therapeutic approach. Success would offer a step change in capability for combating APP, but with far reaching impact for the treatment of bacterial diseases more broadly.

Planned Impact

Please see main proposal.
 
Description Actinobacillus pleuropneumoniae (APP) causes acute and chronic lung disease in pigs and is of substantial economic importance to the worldwide swine industry. There is an urgent need for improved vaccines and therapeutics as antibiotic resistance is an increasing problem. Underpinning this project was our early work showing the importance of small non-coding RNAs (sRNAs) in controlling mRNAs responsible for virulence. Specifically, this grant has enabled the identification of key sRNAs involved in virulence, as well as their associated mRNA targets. This involved bioinformatic analysis of RNAseq data collected under infection and control conditions. The RNA array technology was then used to investigate sRNA-mRNA interactions in high-throughput to explore mRNA target preferences. Biophysical approaches to explore the molecular interactions in more detail were also undertaken, whilst in vivo approaches to confirm suitability as therapeutic targets were also explored. Finally, early steps were made towards identification of a peptide nucleic acid (PNA) to block sRNA functionality for therapeutic effect.

Publications detailing these findings are currently under preparation.
Exploitation Route Applications of the patented RNA array technology:
- This grant provided the first application of the RNA array technology to assessing RNA-interactions identified from bioinformatic analysis of RNAseq data. As such, it demonstrates a strength of coupling these two approaches, highlighting the value of combining the RNA array technology as a high-throughput approach to exploring interactions proposed from RNAseq/bioinformatics data.
- The RNA array technology was also used as a high-throughput approach for testing the efficiency and specificity of PNAs binding to sRNA targets, as well as inhibiting subsequent mRNA interactions. This demonstrates applications of the RNA array in the field of RNA therapeutics, where oligonucleotide mimic molecules (such as PNAs) can be screened for binding to target RNAs, as well as any associated inhibition of subsequent interactions with partner molecules. By analogy, applicability within the synthetic biology domain is also anticipated, where blocking RNA-interactions can bring about a specific transcriptional or translational control effect for the purposes of biotechnology applications.

Applications of the PNA:
- Building on the PNA identified as a potential inhibitor of an sRNA-mRNA interaction important in virulence, steps can be taken to explore its potential as a possible therapeutic.
- Alternatively, the PNA can be used to take a chemical biology approach to 'switching off' a specific sRNA, such that the impact of this can be explored more fully to gain mechanistic understanding.
- More broadly, utilising the PNA to control a specific sRNA-mRNA interaction could be exploited as an RNA switch within a synthetic biology context for biotechnology utilities.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description The findings of this research project strengthen the exemplification of patent applications which have now been granted in the US (US 9,777,268) and Europe (EP 2732047). Additionally, the applications for the RNA technology, demonstrated within the grant, helped secure Innovate UK ICURe funding to explore commercialisation routes, which led to securing a BBSRC Enterprise Fellowship for the PostDoc involved on the project, and a place on the Cambridge-based Start Codon Investment Accelerator programme. This supported steps to create a spin-out venture, RevoNA Bio, which was incorporated in 2022. RevoNA Bio secured an Innovate UK ICURe Follow-on Fund grant and Start Codon investment, and is focused on securing further impact from the RNA technology.
First Year Of Impact 2022
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description Brazil Partnering Award: Imperial-Portsmouth-Vicosa A. pleuropneumoniae collaboration
Amount £50,000 (GBP)
Funding ID BB/S020543/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 05/2023
 
Description University of Portsmouth PhD Studentship (£ 60000; 2016-2019)
Amount £60,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Collaboration with Prof. Denise Bazzolli at the Universidade Federal de Viçosa 
Organisation Federal University of Viçosa
Country Brazil 
Sector Academic/University 
PI Contribution My research team are undertaking molecular interaction studies guided by microbiology data provided by Prof. Bazzolli's team.
Collaborator Contribution Prof. Bazzolli's team are providing microbiology data and expertise.
Impact One of Prof. Bazzolli's team has secured funding to undertake a year-long placement in my research group to support this collaboration.
Start Year 2015
 
Description Collaboration with Prof. Jay Hinton at the University of Liverpool (2017 - 2018) 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Myself and my team provide expertise in the research subject area.
Collaborator Contribution Prof. Hinton provides expertise in bioinformatics to support with data analysis.
Impact The grant and collaboration are on-going.
Start Year 2017
 
Description Collaboration with Prof. Paul Langford at Imperial College London 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team are conducting in vitro molecular interaction studies, and exploring the applicability of utilising our patented novel array technology, to explore cellular mechanisms within bacteria.
Collaborator Contribution Prof. Langford's team provide microbiology expertise and access to in vivo testing studies.
Impact A joint BBSRC grant was secured to support this collaborative research.
Start Year 2015
 
Description Maintaining an Active Online Presence 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My group has an active Twitter account with around 200 followers. We publish highlights from our research, outreach and engagement activities.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016
 
Description Promoting PG study 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Presentation by members of my research team at various departmental events for undergraduates to promote engagement in postgraduate study. this involved the individuals highlighting their research work, including their day to day work, opportunities for collaboration and engagement as well as their outputs and impact.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Science Fairs 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Myself and my team have participated in supporting a number of Science Fairs in the region, engaging with attendees to promote science and the research we undertake.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Translation and commercialisation journey included as a feature in BBSRC's Impact Showcase 2022 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact At the request of BBSRC, myself and the BBSRC Enterprise Fellow working with me on the translation and commercialisation of the RNA technology, were asked to generate a video for the 2022 Impact Showcase. We produced a short video, explaining the journey we had been on, and the role that BBSRC and Innovate UK played in supporting this. The intention was that in sharing our experiences, we would encourage others to explore commercialisation routes for securing impact from their research.
Year(s) Of Engagement Activity 2022
URL https://www.discover.ukri.org/bbsrc-impact-showcase-2022/
 
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Myself and my team regularly support University Open days. Activities can be many and varied, including giving talks, presenting posters, running hands-on laboratory demonstrations and engaging in question and answer sessions. There are usually a number of these events per year, with over 100 participants (schools and college students, sometimes accompanied by a parent/guardian) attending each event. Feedback from such events has highlighted our success in inspiring the next generation of scientists and has been specifically linked to an increase in the number of students applying to study Biochemistry over the last few years.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016
 
Description University of Viscosa visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Visit by a member of my team to the University of Viscosa, Brazil, to explore collaborative working plans going forward.
Year(s) Of Engagement Activity 2020