snoRNA Vectors for Sequence-specific Gene Knockdown in human cells

Lead Research Organisation: University of Dundee
Department Name: Research and Innovation Services

Abstract

Genes carry the genetic instructions to allow cells to build proteins that control chemical reactions within cells and that regulate cell growth and division. Mutations can cause genes to make defective proteins that in turn cause many forms of human disease, including cancer, diabetes and inherited disorders. This proposal will develop a new technology that allows researchers to control which genes are active in human cells and also allows them to inactivate the defective copies of mutated genes and replace them with new genes that can now make the correct and active form of the same protein. This can be widely used to help test and develop new drugs and may also be developed as a direct therapy for use in patients.

Technical Summary

This proposal aims to develop, optimize and evaluate a novel technology for the sequence-specific knock-down and replacement of one or multiple targeted genes in mammalian cells. This will provide a new approach for analysing gene function and for evaluating the effects of chemical inhibitors and drugs in human cells. The technology is based upon our recent discovery of a class of human small nucleolar RNAs (Box C/D snoRNAs) that can be targeted to inhibit the expression of cellular mRNAs and their protein products via the introduction of short regions of sequence complementarity within the snoRNA. This is the subject of a UK patent application entitled ?Targeted Modulation of Gene Expression?. The technology promises major advantages over existing methods, including the ability to knock-down two or more targeted genes simultaneously, thereby overcoming problems associated with analysing biological mechanisms involving genetic redundancy. It also provides a way to rapidly knock down one or more targeted genes and simultaneously replace them with a tagged and/or mutated version of the same gene, using a single vector.
 
Description BBSRC Sparking Impact
Amount £15,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 12/2013
 
Description Analysis of SUMO modified snoRNA Proteins 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Country France 
Sector Academic/University 
PI Contribution We led the collaboration and carried out the major part of the experimental work, including proteomic analysis of nucleoli isolated from cells expressing His-tagged SUMO1 & SUMO2 as well as mutagenesis and transfection studies to characterise the identified SUMO substrates.
Collaborator Contribution Partners carried out specific experiments to analyse the localisation and RNA binding properties of mutant snoRNA proteins created in my laboratory and provided advice and help with several aspects of the project and experimental design. Co-authored manuscripts.
Impact This research led to the publication of Westman et. al (2010) with Dr. Edouard Bertrand.
Start Year 2009
 
Description Bioinformatic analysis of snoRNAs - College of Life Sciences 
Organisation University of Dundee
Department The Barton Group
Country United Kingdom 
Sector Academic/University 
PI Contribution We initiated the collaboration and carried out all of the 'wet lab' experimental work. Our collaborators carried out computational analyses of snoRNA sequences and other bioinformatic studies.
Collaborator Contribution Our collaborators have contributed the bioinformatics to compliment our wet-lab research
Impact There have been three publications published from this work Ono et. al (2011), Ono et. al. (2010) and Scott et. al (2009) with one more paper "Human box C/D snoRNA processing conservation across multiple cell types" Michelle S. Scott1,3*, Motoharu Ono2*, Kayo Yamada2, Akinori Endo2, 4, Geoffrey J. Barton1, Angus I. Lamond2 currently under-review with Nucleic Acids Research
Start Year 2008
 
Description Co-author Sonia Rocha 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Publications
Collaborator Contribution Co-authors
Impact Publications - 23839046 / 23375502 Patents published
Start Year 2006
 
Description Human miRNA precursors with box H/ACA snoRNA features 
Organisation University of Dundee
Department Biological Chemistry and Drug Discovery (BCDD)
Country United Kingdom 
Sector Academic/University 
PI Contribution Bioinformatic analysis was done by Michelle Scott and then experiments were performed in the Lamond lab.
Collaborator Contribution Bioinformatic analysis carried out by the Barton Group. For their hypothesis experimental work was undertaken.
Impact Publication: Pubmed id 19763159
Start Year 2009
 
Description co-author Geoff Barton 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Publication
Collaborator Contribution co-author
Impact PMID: 19763159 PMID: 20219969 PMID: 20663773 PMID: 20826608 PMID: 21247878 PMID: 21272300 PMID: 21937730 PMID: 22199253
Start Year 2009
 
Description co-author Sonia Rocha 
Organisation Wellcome Trust
Department Wellcome Trust Centre for Gene Regulation and Expression
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Publications
Collaborator Contribution Co-authors
Impact Enhanced snoMEN vectors facilitate establishment of GFP-HIF-1a protein replacement human cell lines - to be published in 2016
Start Year 2015
 
Title TARGETED MODULATION OF GENE EXPRESSION 
Description The present invention relates to a method of modulating gene expression using snoRNA molecules or snoRNA like molecules or fragments, designed to target specific nucleic acid sequences. 
IP Reference WO2009037490 
Protection Patent application published
Year Protection Granted 2009
Licensed No
Impact n/a
 
Title TARGETING OF MIRNA PRECURSORS 
Description The present invention relates to a method of targeting mi RNA and/or premiRNA molecules in order to treat diseases that are linked with mi RNA expression, such as certain cancers. The present invention also provides modified sno RNA molecules for targeting mi RNA molecules for use in treating diseases that are linked with mi RNA expression, such as certain cancers. 
IP Reference US2014275221 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact N/A
 
Company Name Dundee Cell Products 
Description Dundee Cell Products (DCP) is a spin out company of University of Dundee of which Angus I Lamond is a co-founder. http://www.dundeecellproducts.com/ ; http://www.dundeecellproducts.com 
Year Established 2006 
Impact 1. Dundee Cell Products has successfully commercialised SILAC quantitative proteomics technology to academic and commercial customers. It has carried out SILAC analysis for customers in about six universities and recently gained a service contract with one of the world's largest pharmaceutical companies to apply this technology in their drug discovery and development process. The company has also developed various SILAC ready-to-use tissue culture media that facilitate the culture of cell lines for subsequent quantitative proteomics analysis. These special SILAC media have gained customers all over Europe and in North America. 2. Since spinout, the company has commercialised life sciences research reagents including antibodies, recombinant proteins, DNA clones, stable cell lines, etc., many of which have been developed or licensed from Universities/academic institutions. 3. The company has created new jobs by employing five highly skilled staff (3 post-doctoral, two undergraduates). It also provides part-time work to two contractors (bookkeeper and accountant), work to local businesses and outsources some work to the University of Dundee thus providing extra revenue to the institution.
Website http://www.dundeecellproducts.com/
 
Description GRE videos 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The GRE video is available on our website for anyone to view

Positive feedback
Year(s) Of Engagement Activity 2008
 
Description Invited Seminar Speaker 
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 Presentations of results

Regularly invited as a seminar speaker
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013
 
Description Member of Editorial Board - Science Scotland 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
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Results and Impact Science Scotland is availalble online and is distributed nationwide

Recognised for contribution to Science and asked to contribute an editorial piece
Year(s) Of Engagement Activity 2012
 
Description Open Doors Day (Oranges & Lemons Nursery, Dundee) 
Form Of Engagement Activity Participation in an activity, workshop or similar
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Geographic Reach Local
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Results and Impact Participation in scientific exhibition activity, workshop at this local nursery. Targeted at Primary school children/parents.
Year(s) Of Engagement Activity 2016
 
Description Open Doors Day at the College of Life Sciences 
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Geographic Reach Local
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Results and Impact Approx 150-180 members of the public visit the Open Doors Days each year to take part in tours, see exhibits and to ask researchers questions.

The number of visitors to Open Doors Days has been steadily increasing over the last few years
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014
 
Description School Visit - Fife 
Form Of Engagement Activity Participation in an activity, workshop or similar
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Geographic Reach Local
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Results and Impact Presentation of research to school children

Positive feedback from schools and being invited back
Year(s) Of Engagement Activity 2006,2007,2008,2009,2016
 
Description WT 75th Anniversary Celebrations (Dundee 2011) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact HIghlighted the good work of the WT, London by hosting this celebration

UoD were asked to host this celebration
Year(s) Of Engagement Activity 2011
 
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HIghlighting the opportunities available in science
Year(s) Of Engagement Activity 2011,2012