BioMatNet Item: QLK3-2001-00448 - Development of highly specific enzymes for genome manipulation (EnGeM)

[BioMatNet Database - FP5 Quality of Life Programme]

QLK3-2001-00448
Development of highly specific enzymes for genome manipulation (EnGeM)

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Biotechnology : Fine Chemicals : Quality of Life - 3. The Cell Factory

Type of Project	Research Project
Contract No	QLK3-2001-00448
Total Cost
EC Contribution	2,629,242 EUR
Start Date	01-01-2002
Duration	36 Months

Abstract

Efficient exploitation of genome information will, among others, depend on our ability to sequence/specifically modify genomic DNA in vivo. A consortium of eight academic and three industrial units will engineer endonucleases and DNA methyltransferases of programmable specificities which can cleave or methylate a single site in DNA as complex as the human genome. The ability of the enzymes to induce gene replacement or gene silencing by DNA cleavage or cytosine-methylation, respectively, at predetermined sites, will be explored. The project involves coupling of oligonucleotides (ODNs) and peptide nucleic acids (PNAs) to the N-, or C-termini of the C5-methyltransferase M.SssI and a single-chain derivative of the restriction endonuclease PvuII. The covalently attached ODNs and PNAs will serve as affinity tags to direct the enzyme to specific sequences in the genome. Methods will be developed to introduce the enzymes into cells. Conditions for avoiding enzyme action at nonspecific sites will be explored. Development of enzymes of such high specificity should greatly enhance our ability to manipulate genomes and may lead to medical applications e.g in gene therapy.

Objectives

Construction of variants of the C5-methyltransferase M.SssI and of a single-chain derivative of the restriction endonuclease R.PvuII (sc-R.PvuII) which are suitable for terminal coupling to oligonucleotides (ODN) or peptide nucleic acids (PNA). Coupling of M.SssI and sc-R.PvuII to ODNs and PNAs by three different methods. Biochemical, biophysical and crystallographic studies of the affinity-tagged enzymes with emphasis on substrate specificity. Introduction of the conjugates into cells and testing their actions in vivo using methods developed by the industrial partners. Testing the potential of targeted DNA cleavage by R.PvuII-ODN/DNA conjugates to replace defective genes and the potential of targeted cytosine methylation by M.SssI-ODN/PNA conjugates to influence transcription of targeted genes. Strengthening European industrial competitiveness by involving three European companies in the development, testing and commercialisation of biomedical applications and by disseminating results to potential European users.

Activities

Since engineering of restriction endonucleases and DNA methyltransferases based on so-called rational approaches turned out to be extremely difficult for the development of DNA modifying enzymes with programmable specificities, chemical methods which allow specific coupling of enzymes with ODNs or PNAs capable of forming a triple helix on DNA will be used. Because triple helix formation is highly specific, the ODN/PNA moiety can target the DNA modifying enzymes used in the project as model systems to particular sites. Enzyme specificity can be programmed by coupling to different ODN or PNA sequences. An engineered, monomeric variant of the restriction endonuclease R.PvuII (it represents a technology that is exclusively available in the consortium) and the DNA methyltransferase M.SssI will be used as model systems. Mutants of these enzymes, which are suitable for coupling with ODN and PNA and which have been optimised for DNA-binding will be produced and purified in large quantities.

Three different coupling methods to ODN or PNA affinity tags will be explored:

classical coupling using heterobifunctional crosslinking reagents.
native chemical peptide ligation (NCPL).
intein-mediated protein ligation (IPL).

The biochemical, biophysical and structural characterisation of the interactions of promising enzyme-ODN/PNA conjugates with DNA will be carried out and will form the basis for the redesign and improvement of the conjugates' properties. A cellular delivery system developed by one of the industrial partners will be used to target the enzyme-ODN/PNA conjugates to appropriate cells and study their actions in vivo. The potential of targeted cytosine methylation through M.SsI-ODN/PNA conjugates to influence (silence) the transcription of targeted genes (particularly genes involved in disease) will be tested and the potential applicability for diagnostic/therapeutic purposes will be explored. One industrial partner will perform the large-scale production of promising enzymes. An efficient mechanism for monitoring of progress, the exploitation of results and the information dissemination has been established.

Deliverables

production and purification in large quantities of functional sc-R.PvuII and M.SssI molecules and their engineered derivatives.
protocols for coupling of sc-R.PvuII and M.SssI to ODN or PNA.
structural, biochemical, kinetic, thermodynamic characterisation of the interactions of the conjugates with DNA.
protocols to deliver conjugates to cells.
protocols for targeted gene silencing in cells using M.SssI variant - ODN/PNA fusion constructs.
concept for further experiments to establish biomedical applications.
dissemination of information.

Contacts

Coordinator

M. KOKKINIDIS / Foundation for Research and Technology Hellas Institute of Molecular Biology and Biotechnology / GREECE

Participant