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Study of Prion Proteins using Relevant Aptamers

#2750


Study of Prion Proteins using Relevant Aptamers, Obtained by SELEX, Aimed to Develop New Approaches in Diagnostics and Therapy of Prion-Mediated Diseases

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-DID/Diagnostics & Devices/Medicine

Status
8 Project completed

Registration date
07.04.2003

Completion date
04.06.2009

Senior Project Manager
Weaver L M

Leading Institute
State Scientific Center of Genetics and Selection of Industrial Microorganisms (GosNIIGenetica), Russia, Moscow

Supporting institutes

  • Russian Cardiology Research Center / Institute of Experimental Cardiology, Russia, Moscow\nInstitute of Immunological Engineering, Russia, Moscow reg., Lyubuchany\nVIEV (Experimental Veterinary), Russia, Moscow

Collaborators

  • University of Kent at Canterbury, UK, Canterbury\nUniversity of Illinois, USA, IL, Chicago\nATF Advanced Technologies & Fuels Canada, Inc., Canada, ON, Ottawa

Project summary

Prions constitute a special class of protein infectious agents which cause induced incurable diseases of the central nervous system in human and animals.

At present no methods for the treatment of prion diseases exist, and the problem of the development of effective therapeutic and diagnostic techniques of prion diseases is considered to be the task of priority taking into account the predictions of the possible considerable epidemic of account the predictions not excluding the considerable epidemic of “new variant” Creutzfeld–Jacob disease for the nearest 10-15 years. The perspective way of treatment of prion diseases is considered to be the prevention of conversion of wild type protein into its prion form. Such methods can be developed by use of experimental systems based on yeast.

The available diagnostic techniques for prion diseases based exclusively on the study of post-mortem samples of brain tissues, appear to have some serious shortcomings. The methods based on the study of infectivity of brain tissue samples from seek animal are time consuming. The obtaining of specific antibodies with high affinity necessary for immunodetection is connected with considerable difficulties because prions are very close to normal PrP proteins by their antigen structure.

A good alternative to the existing approaches for diagnosis of prion diseases as well as for creation of tools for their treatment, appeared to be the procedure of construction and selection of short oligonucleotides of DNA or RNA origin carried out from an enormous set of initial mixture of synthesized fragments with randomized sequence ("SELEX" – Systematic Evolution of Ligands by Exponential enrichment).Aptamers specific to the pathogenic isoform of prion proteins have not still been obtained. Availability of such aptamers for the researchers would provide the beginning of the development of techniques for detection and analysis of infectious prion proteins in samples obtained from animals. This would be a base for the development of lifetime preclinical methods of early diagnosis of prion diseases.

Yeast prions, and Sup35 prion protein, in particular, represent a highly effective model system for the development of techniques based on aptamers for diagnosis and prevention of conversion of a normal PrPC into infectious PrPSc form. The procedure of SELEX to be developed in the Project will be used for selection of aptamers able to bind infectious prion protein PrP which is responsible for the development of bovine transmissible spongiform encephalopathy.

The aim of the Project:


- obtaining of aptamers to the prion form of the Sup35 protein, selection of the aptamers which bind the infectious prion protein PrP and investigation of prion forms of proteins by means of aptamers obtained;
- demonstration of possibility for diagnosis using aptamers of protein prion forms both in yeast and in animal samples;
- study of the possibility of inhibition of prionization of wild type Sup35 protein using the obtained aptamers.

The Project is based on the expertise of the researches and their achievements made previously during their work on the ISTC Project #1038 when various modifications of SELEX performance were mastered and aptamers to IL-8 were obtained.

The authors of the Project were the first to study the structural-functional organization of the Sup35 protein and the functional role of the protein domains. The investigations carried out have clearly demonstrated the prion-like nature of the Sup35 protein, which was also demonstrated in studies of its conversion into a prion form in vitro. The Project performers have got a great experience both in the construction of strains-producers of recombinant proteins and in structural-functional studies of recombinant proteins. The Scientific staff of the Institute of Experimental Cardiology, Russian Cardiology Research-Industrial Center, Ministry of Public Health of the Russian Federation (Institute of Cardiology) have essential achievements in the field of research of prions in yeast (Saccharomyces cerevisiae). Particularly, a model system has been developed lately based on the Sup35 protein conversion into a prion form. A great variety of yeast strains and transforming vectors, anti Sup35 antibodies are available. A great number of factors controlling the efficiency of protein conversion into prion form has been identified.

All-Russian Ja. R. Kovalenko Institute of Experimental Veterinary Science (VIEV ), Russian Academy of Agricultural Sciences possesses some strains of animal pathogenic prions – sheep scrapie and bovine transmissible spongiform encephalopathy. The scientific staff of the Institute has experience in experimental reproduction of these diseases in naturally sensitive animals, they have pathologic samples from sick animals and have experience in isolation and purification of the agents (pathogenic prions of sheep scrapie and bovine spongiform encephalopathy), and in immunohystochemical detection of prion protein in pathological material. The Institute researchers got to know the techniques of the work with animal pathogenic prions at the Central Veterinary Laboratory of England (Waybridge) and at the Division of Human and Animal Neuropathogenic Infections of the Institute of Animal Health (Edinburg) in 1990 and in 1992, and at the Ireland Veterinary Research Laboratory (Dublin) in 1997.

The level of studies was confirmed by more than 16 publications made by the authors of Project in international reviewed journals (Science, Mol.Cell.Biol., Biochim Biophys Acta, etc.).

Expected Results of theoretical importance.

Resulting from the SELEX procedure, aptamers efficiently binding with prion forms of proteins will be obtained.

New data based on their interactions will be obtained:


- on protein sites important for prionization,
- on the levels of similarity and differences of the spatial structure of prion proteins of lower eukaryotes and animals,
- on the level of similarity and differences of the structure of prion-like amyloids obtained in vitro, with prion aggregates accumulating in cells,
- on primary and spatial structure of aptamers to polymer forms of prion proteins,
- on peculiarities of prion protein aggregation.

The data obtained would enable the development of new approaches in study of prion infections.

Results of applied value.

During the work under the Project, SELEX procedure for obtaining aptamers specific to the proteins in the prion form will be elaborated. Application of such aptamers can be demonstrated for development of rapid and efficient diagnosis of prion diseases of men and animals. Obtaining of aptamers able to inhibit prionization of the normal forms of a protein would enable development of new approaches to the therapy of prion diseases.

Results of commercial value.

The aptamers are planned to be obtained and the results of the analysis of their interaction with prion proteins could be patented and become a base for the development of pioneer diagnostic kits and therapeutic means against prion diseases. The Project team has conditions for their construction and testing in practice.

The importance of the project is confirmed by the fact that the project attracts attention of foreign scientists who are willing to participate in joint projects. Realisation of the suggested project will contribute significantly to fundamental and applied biology, immunology and medicine and would finally contribute to development of pharmaceutical preparations of a new generation. It will also help to solve a social problem, i.e. permit the scientists and engineers who earlier worked on state orders of the Ministry of Defence to reorient their research interests. They would be able to apply their experience in basic and applied studies and thus contribute to resolution of international scientific and technological problems in biology, medicine and veterinary. This would create long-term perspectives for fruitful activity of the international research community.

Project provides:

- construction of special systems of production and isolation of native and prion forms of Sup35 protein,


- elaboration of purified prion proteins from animal samples with bovine spongiform encephalopathy,
- obtaining of aptamers to prion form of the Sup35 protein and animal prion proteins,
- biochemical and physico-chemical studies of the interaction of aptamers with prion and normal forms of the Sup35 protein,
- study of inhibition of conversion of the Sup35 protein into a prion form by selected aptamers,
- study of binding of aptamers to prion aggregates of the Sup35 protein isolated from different [PSI+] yeast strains,
- study of the binding of obtained aptamers with PrP protein purified from animals with bovine spongiform encephalopathy.


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