Title of project or programme

Molecular diagnostic strategies in prion disease

Principal Investigators of project/programme grant
Title Forname Surname Institution Country
Dr Graham Jackson MRC Prion Unit UK
Address of institution of lead PI
Institution MRC Prion Unit
Street Address Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square
City London
Postcode WC1N 3BG
Country
  • United Kingdom
Source of funding information

Medical Research Council

Total sum awarded (Euro)

3733703.00

Start date of award

01-04-2005

Total duration of award in months

60

The project/programme is most relevant to
  • Prion disease
Keywords
Research abstract in English

The emergence of vCJD, and the experimental confirmation that it is caused by the same prion strain as bovine spongiform encephalopathy (BSE) has dramatically highlighted the need for ante-mortem diagnostic tests for prion disease and reliable methods for the destruction of prion infectivity. The extremely prolonged and variable incubation periods of these diseases, particularly when crossing a species barrier, and the demonstration of a discrete number of as yet uncharacterised genetic loci with a major effect on incubation periods, means that it will be some years before the parameters of any human epidemic can be predicted with confidence. In the meantime, we are faced with the possibility that significant numbers in the population may be incubating this disease and infect others via blood transfusion, blood products, tissue and organ transplantation and other iatrogenic routes. The recent demonstration of sub-clinical carrier states of prion infection in animal models is also relevant to public health, both with respect to prion zoonoses and iatrogenic transmission of human prions. The immediate solution to many of these problems is to provide a sensitive blood-based pre-symptomatic diagnostic test for prion infection. This programme aims to investigate basic aspects of prion biology that are directly relevant to methods for blood-based diagnostic testing, and to couple such basic research with translational research such as immunoassay optimisation and proteomics screening in order to provide a co-ordinated approach to ante-mortem diagnostics.

Key projects within the research programme are aimed at developing methods for the amplification of PrPSc and infectivity from patient blood samples. The nascent technology of PMCA (Protein Misfolding by Cyclic Amplification) is currently being investigated as a means to amplify PrPSc to detectable levels from normal blood samples spiked with high dilutions of infected brain homogenate. If successful this method will be used to amplify material from a longitudinal study in wild-type mice that has provided blood samples from various points throughout the incubation period of disease and ultimately in blood samples obtained from vCJD patients. In parallel, attempts are being made to ‘seed’ reactions in which recombinant PrP can be induced to fibrilise. Such reactions can be seeded and accelerated with preformed fibrils of recombinant PrP and if they can be also be catalysed by the addition of infected tissue homogenates or blood the incorporation of appropriately labelled recombinant PrP into fibrils could be sensitively detected by FRET.

Separate projects are underway to increase the sensitivity of PrPSc detection in immunoassays by developing new methods for the enrichment of disease-associated PrP prior to assay and by improving sandwich ELISA formats by screening a panel of over 80 monoclonal anti-PrP antibodies for optimum pairings.

Lay Summary

    Principal Investigators

    Dr G S Jackson

    Institution

    MRC Prion Unit

    Contact information of lead PI

    Country

    United Kingdom

    Title of project or programme

    Molecular diagnostic strategies in prion disease

    Source of funding information

    MRC

    Total sum awarded (Euro)

    € 5,091,026

    Start date of award

    01/04/2011

    Total duration of award in years

    5.0

    The project/programme is most relevant to:

    Prion disease

    Keywords

    Research Abstract

    The emergence of vCJD, and the experimental confirmation that it is caused by the same prion strain as bovine spongiform encephalopathy (BSE) has dramatically highlighted the need for ante-mortem diagnostic tests for prion disease and reliable methods for the destruction of prion infectivity. The extremely prolonged and variable incubation periods of these diseases, particularly when crossing a species barrier, and the demonstration of a discrete number of as yet uncharacterised genetic loci with a major effect on incubation periods, means that it will be some years before the parameters of any human epidemic can be predicted with confidence. In the meantime, we are faced with the possibility that significant numbers in the population may be incubating this disease and infect others via blood transfusion, blood products, tissue and organ transplantation and other iatrogenic routes. The recent demonstration of sub-clinical carrier states of prion infection in animal models is also relevant to public health, both with respect to prion zoonoses and iatrogenic transmission of human prions. The immediate solution to many of these problems is to provide a sensitive blood-based pre-symptomatic diagnostic test for prion infection.||This programme aims to investigate basic aspects of prion Biology that are directly relevant to methods for blood-based diagnostic testing, and to couple such basic research with translational research such as immunoassay optimisation and proteomics screening in order to provide a co-ordinated approach to ante-mortem diagnostics.||key projects within the research programme are aimed at developing methods for the amplification of PrPSc and infectivity from patient blood samples. The nascent technology of PMCA (Protein Misfolding by cyclic Amplification) has been investigated as a means to replicate PrPSc to detectable levels from normal blood samples spiked with high dilutions of infected brain homogenate and from blood sampled from mice in the pre-clinical stages of experimental prion disease. We aim to dissect these reactions to define and understand the molecular events that constitute prion replication and identify what components of PMCA substrate (currently treated brain homogenate) are crucial for efficient prion replication. In parallel simplified amplification reactions in which recombinant PrP can be induced to fibrilise are being studied. Such reactions can be seeded and accelerated with preformed fibrils of recombinant PrP. These reactions can be also be catalysed by the addition of infected tissue homogenates or blood. We are investigated means to enhance the speed and sensitivity of such assays and aim to combine knowledge gained from understanding PMCA with recombinant PrP to enable purely synthetic, in vitro prion replication.||Separate projects are underway to increase the sensitivity of PrPSc detection in immunoassays by developing new methods for the capture and enrichment of disease-associated PrP prior to assay and by improving sandwich ELISA formats by screening a panel of over 80 monoclonal anti-PrP antibodies for optimum pairings. We have made considerable progress and extended the sensitivity of immunodetection to levels greater than the sensitivity afforded by rodent bioassays, being able to detect infected vCJD brain homogenate at a dilution of 1010. Application of this methodology to both endogenous rodent and patient blood samples has confirmed that we are able to differentiate infected blood from control samples.

    Lay Summary

    The majority of the UK population and a lesser proportion of continental European and the rest of the world have been exposed to BSE contaminated foods and this has resulted in the new disease vCJD. Although the number of patients remains small, the number of people infected but without any signs of illness is unknown and could be very large. The time from being infected to showing signs of the disease can be very long and maybe up to 50 years. During this period there is a risk of spreading the disease through the contamination of medical and dental instruments; the use of contaminated blood for transfusion and the transplantation of infected organs such as kidneYess. All of these dangers can be avoided if simply tests are developed that can detect CJD. Accurate tests that work on blood samples would allow the testing of everyone who donated blood for transfusions and the testing of anyone due to have surgery.||After infection with BSE there is a characteristic change that occurs to one of the bodys normal proteins in people who have the disease and the detection of this altered protein or prion can be used to show someone has CJD. Unfortunately the amounts of prion are very low in blood and are difficult to tell apart from the large amounts of the normal protein that are always present in both healthy and infected people.||We are trying different ways of detecting this rare altered protein. One approach is to copy the way the prion reproduces itself in the human body during disease. This will allow us to amplify the very small amounts of prion in blood samples up into large amounts that can be easily detected using ordinary laboratory tests.||As an alternative to amplification we are also finding ways to concentrate prions. If we can find ways to extract all the prions from a test-tube of blood this will make their detection much easier. We have found ways to extract prions from blood and detect them sufficiently well to distinguish infected blood from normal samples. We are trying to understand how prions copy themselves in replication reactions and aim to use concentration and replication to make prions in blood easily detectable.

    Further information available at:

Types: Investments > €500k
Member States: United Kingdom
Diseases: Prion disease
Years: 2011
Database Categories: N/A
Database Tags: N/A

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