Applications of synthetic nucleic acids and their peptide conjugates

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Most clinically used drugs are small molecules targeted to inhibit the action of an essential protein. A new approach in drug development is to target the genetic material in a cell (nucleic acid) that codes for such proteins, of which the most readily accessible is ribonucleic acid (RNA). Short pieces of synthetic nucleic acid (oligonucleotides, ONs) are used to form complementary base pairs with RNA inside cells (antisense) so as to block its ability to express a protein or carry out a regulatory function. For therapeutic use, ONs are chemically modified to prevent degradation by enzymes in blood and in cells and to slow clearance from the body. Modified ONs are being used clinically and are in clinical trials for treatment of viral infections, obesity, inflammation, cancer and genetic diseases. One problem is that ONs do not enter most human cells efficiently. Our research involves chemical synthesis of ON analogues and their peptide conjugates designed to improve cell uptake and RNA targeting efficiency as well as efficacy in mouse models of disease in partnership with collaborators in Oxford. Particular effort is focussed on peptide nucleic acids (PNA) and phosphorodiamidate morpholino ONs (PMO), ON analogues that are charge neutral. PNA and PMO ON types bind strongly to complementary RNA with high sequence specificity. We attach to PNA and PMO short novel peptides (protein segments) that have cell penetrating properties. We are applying such antisense ONs and their peptide conjugates to redirect pre-messenger RNA splicing in the nucleus of cells. We focus in particular on developing ON-based strategies towards developing new therapeutic leads for treatment of hitherto incurable neuromuscular genetic diseases, such as Duchenne muscular dystrophy, spinal muscular atrophy and myotonic dystrophy. In conjunction with colleagues at Oxford University we hope to take lead peptide-PMO conjugates towards clinical trials in one or more of these diseases.

Technical Summary

Short synthetic oligonucleotides (ONs) and their analogues are used extensively to inhibit gene expression in cells and as potential therapeutics. ONs bind complementary RNA and either sterically block an essential RNA function or induce the cleavage of the RNA by a cellular enzyme RNase H. ONs must be able to penetrate the desired cells (in culture or in vivo), reach their RNA targets in the cytosol or nucleus, and bind specifically to the correct RNA site. We utilize ONs that carry modifications to help RNA binding and to resist degradation by cellular and serum nucleases. However, ONs are generally poor in cell penetration. Our research focuses on the chemical synthesis of ON analogues as conjugates with synthetic peptides that show enhanced cell delivery properties in the absence of additional carriers (e.g. cationic lipids, nanoparticles). A major part of current work focuses on peptide conjugates of charge-neutral ON analogues, peptide nucleic acids (PNA) and phosphorodiamidate morpholino oligonucleotides (PMO). We designed a novel class of cell penetrating peptides (CPPs) called Pip that have increased serum stability and when linked to PNA or PMO act as steric blocking agents to redirect splicing of pre-mRNA with excellent efficiencies. We have used Pip conjugates of PMO ONs targeting exon 23 of the dystrophin gene in a mdx mouse model of Duchenne Muscular Dystrophy (DMD). In collaboration with the group of Matthew Wood in Oxford, we obtained substantially enhanced exon skipping and dystrophin production in vivo compared to naked PMO. We have since developed further peptide classes as PMO conjugates with high activity in the mdx mouse model that are suitable as clinical leads for treatment of DMD patients. This work has been extended to obtain high activity for novel peptide conjugates of PMO in mouse models of other neuromuscular diseases, namely spinal muscular atrophy (SMA) and myotonic dystrophy. We hope to exploit these leads through establishment of a joint MRC-Oxford spln-off company.

Publications

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Abes R (2011) Splice redirection as a convenient assay to monitor CPP-ON efficiency and mechanism. in Methods in molecular biology (Clifton, N.J.)

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Abes R (2007) Cell-penetrating-peptide-based delivery of oligonucleotides: an overview. in Biochemical Society transactions

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Abes R (2008) Arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides. in Journal of peptide science : an official publication of the European Peptide Society

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Abes S (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. in Journal of controlled release : official journal of the Controlled Release Society

 
Description AFM Research Project
Amount € 279,754 (EUR)
Funding ID AVRUBKO 
Organisation French Muscular Dystrophy Association (AFM) 
Sector Charity/Non Profit
Country France
Start 04/2011 
End 03/2015
 
Description AFM Research Project
Amount € 198,658 (EUR)
Organisation French Muscular Dystrophy Association (AFM) 
Sector Charity/Non Profit
Country France
Start 04/2014 
End 03/2016
 
Description Action Duchenne
Amount £25,000 (GBP)
Organisation Action Duchenne 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2008 
End 07/2010
 
Description AstraZeneca/LMB Blue skies Initiative
Amount £73,799 (GBP)
Organisation AstraZeneca 
Department Research and Development AstraZeneca
Sector Private
Country United Kingdom
Start 09/2015 
End 08/2016
 
Description Cesar Milstein Studentship
Amount £75,000 (GBP)
Organisation British Society for the History of Science (BSHS) 
Department Darwin Trust of Edinburgh
Sector Charity/Non Profit
Country United Kingdom
Start 01/2008 
End 12/2010
 
Description DoH/WT Health Innovation Challenge Fund
Amount £175,234 (GBP)
Funding ID R15741/DPAG/Wood 
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 03/2011 
End 02/2014
 
Description EC FP5 shared costs
Amount £243,425 (GBP)
Organisation European Commission 
Department Fifth Framework Programme (FP5)
Sector Public
Country European Union (EU)
Start 04/2003 
End 03/2006
 
Description LMB/AztraZeneca Blue Skies Initiative
Amount £143,104 (GBP)
Funding ID BSF14 
Organisation AstraZeneca 
Department Research and Development AstraZeneca
Sector Private
Country United Kingdom
Start 01/2017 
End 09/2017
 
Description MRC DPFS
Amount £188,183 (GBP)
Funding ID MR/L013142/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description MRC Development Gap Funding
Amount £180,848 (GBP)
Funding ID A853-0132 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2011 
End 01/2014
 
Description MRCT Development Gap Funding
Amount £65,153 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 08/2008 
End 12/2009
 
Description NHMRC Early Career Fellowship
Amount $313,668 (AUD)
Organisation National Health and Medical Research Council 
Sector Public
Country Australia
Start 08/2012 
End 07/2014
 
Description Royal Society Award-Tandon
Amount £3,982 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2007 
End 12/2007
 
Description University of Oxford/Duchenne UK
Amount £50,000 (GBP)
Organisation Duchenne UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 12/2017
 
Description Wellcome Trust Collaborative Projects
Amount £22,651 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 12/2012
 
Description Astra-Zeneca/LMB joint grant 
Organisation AstraZeneca
Department MedImmune
Country United Kingdom 
Sector Private 
PI Contribution Blue-skies research project providing 9 months salary and overheads to
Collaborator Contribution Provision of antibodies
Impact Chemistry and cell testing from LMB. Antibodies from Medimmune.
Start Year 2015
 
Description CRB License and collaboration 
Organisation Cambridge Research Biochemicals
Country United Kingdom 
Sector Private 
PI Contribution Development of synthesis method for preparation of libraries of peptide conjugates of biocargoes
Collaborator Contribution Licensing of patent application and development of synthesis service for customers. Patent application dropped.
Impact Patent application was not continued
Start Year 2013
 
Description Development of PNA Synthesizer 
Organisation Queen's University Belfast
Department Centre for Experimental Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Initial discussion about collaboration was followed by agreement to collaborate
Collaborator Contribution co-development of equipment for PNA synthesis
Impact Co-development of equipment and method for PNA synthesis. Paper to be submitted Dec 2009
Start Year 2008
 
Description EU COST programme BM 1207 
Organisation European Cooperation in Science and Technology (COST)
Country Belgium 
Sector Public 
PI Contribution Presentations and discussions to a large range of EU research groups involved in exon skipping of pre-mRNA
Collaborator Contribution Presentations and discussions
Impact multi-disciplinary collaboration
Start Year 2013
 
Description EU FP5 Consortium 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Part of EU FP5 consortium.
Collaborator Contribution Exchange of ideas, materials and joint publications. Consultancy
Impact Exchange of ideas, materials and joint publications.
Start Year 2006
 
Description EU FP5 Consortium 
Organisation Panagene
Country Korea, Republic of 
Sector Private 
PI Contribution Part of EU FP5 consortium.
Collaborator Contribution Exchange of ideas, materials and joint publications. Consultancy
Impact Exchange of ideas, materials and joint publications.
Start Year 2006
 
Description MDEX clinical consortium 
Organisation University College London
Department Institute of Child Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint work and grant applications on Duchenne muscular dystrophy
Collaborator Contribution Grants awarded Wellcome/AFM
Impact Grants awarded from the Wellcome Trust and the AFM began 2011. Now retired from the MDEX partnership.
Start Year 2010
 
Description Peptide synthesizer 
Organisation Activotec SPP
Country United Kingdom 
Sector Private 
PI Contribution Development of Activotec peptide synthesizer with help from staff member David Owen
Collaborator Contribution Some initial collaboration in developing new peptide synthesiser and short term evaluation. project terminated
Impact Some initial collaboration in developing new peptide synthesiser and short term evaluation. project terminated
Start Year 2006
 
Description Sample Materials from Link 
Organisation Link Technologies
Country United States 
Sector Private 
PI Contribution MRC license income from patent assignment.
Collaborator Contribution Receipt of sample materials for oligonucleotide and PNA synthesis
Impact Receipt of sample materials for oligonucleotide and PNA synthesis
Start Year 2006
 
Description SiRNA testing 
Organisation Alnylam Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Received test cell line from Alnylam for siRNA testing. Used and reported in talks, but not published in detail
Collaborator Contribution Received test cell line from Alnylam for siRNA testing. Used and reported in talks, but not published in detail
Impact Material Transfer Agreement (MTA) - Double luciferase test cell line
Start Year 2006
 
Title Cell Penetrating Peptide 
Description A novel design for a cell penetrating peptide for conjugation to biologically active cargoes such as oligonucleotides and peptides. Particular applications shown to conjugation to charge-neutral PNA and PMO oligonucleotides 
IP Reference US8575305 
Protection Patent granted
Year Protection Granted 2013
Licensed Yes
Impact Peptide sequences known as Pip that can be used as a delivery system for transporting peptides or nucleic acids into cells. Original patent application applied for June 2008, issued Dec 2009. Non exclusive licensing to Shire Pharmaceuticals. Other licenses have been explored.
 
Title Cell Penetrating Peptides having a central hydrophobic domain 
Description A second series of cell penetrating peptides known as Pip with activity in muscle and heart as conjugates of PMO oligonucleotides 
IP Reference US9302014 
Protection Patent granted
Year Protection Granted 2016
Licensed Yes
Impact License includes reporting of data by licensee on toxicology of Pip-PMO in rats. Additional licenses have also been explored.
 
Title Cell penetrating peptides modified by glycosylation 
Description Cell penetrating peptides modified by glycosylation for brain entry for treatment of spinal muscular atrophy. 
IP Reference WO2018150196 
Protection Patent application published
Year Protection Granted 2018
Licensed Yes
Impact Not as yet had further impacts
 
Company Name Sophos Therapeutics 
Description As yet a paper company ready to exploit cell penetrating peptides for DMD and other diseases; NULL 
Year Established 2008 
Impact The company was disbanded in 2011 as it was not regarded as serving a useful purpose
 
Description Action Duchenne Conference 2008 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Gave a lecture about my research

Funding received from Action Duchenne towards research
Year(s) Of Engagement Activity 2008
 
Description Action Duchenne Conference 2009 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact gave a lecture

further funding from Action Duchenne
Year(s) Of Engagement Activity 2009
 
Description Action Duchenne Conference 2011 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact gave talk

company interest in our work for possible collaboration
Year(s) Of Engagement Activity 2011
 
Description Action Duchenne Conference 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Interest in our research from funders, companies and patient groups

Awareness of our pre-clinical and clinical collaborative drug development programmes
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013
 
Description Action Duchenne conference 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact gave talk

further funding opportunities
Year(s) Of Engagement Activity 2010
 
Description Dutch Undergraduates visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Visit of 6 students from Radboud Honours Academy, Radboud University, The Netherlands, who were studying muscular dystrophies. Gave them a talk about research careers in the MRC.

Interest from one student in potential research
Year(s) Of Engagement Activity 2011
 
Description Interview BBC Radio Camb 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact 6 min radio interview BBC radio Cambridgeshire 07.45 regarding my comments on a scientific paper from another lab and mentioning our own work in relation

No further outcome
Year(s) Of Engagement Activity 2008
 
Description LMB alumni symposium 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Many visits by alumni to our research group and viewing of poster

greater awareness of research of group
Year(s) Of Engagement Activity 2014
 
Description Science Media Centre Press briefing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact 10 min talk to journalists about recent developments in therapeutics of RNA interference

no further interviews took place but name more widely known
Year(s) Of Engagement Activity 2008
 
Description Visit Swedish PhD Group 
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 Visit of about 20 Swedish Ph D students to LMB to see what is involved in postdoctoral research

Potential applicants for future postdoc positions
Year(s) Of Engagement Activity 2010