The development of candidate therapeutics for Transmissible Spongiform Encephalopathies.
Lead Research Organisation:
University of Nottingham
Department Name: School of Veterinary Medicine and Sci
Abstract
Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of progressive, fatal neurodegenerative disorders with no treatment or cure. Among this group are both human (eg. Creutzfeldt-Jakob disease - CJD, fatal familial insomnia - FFI, Gerstmann-Sträussler-Scheinker syndrome - GSS) and animal diseases (eg. scrapie, bovine spongiform encephalopathy - BSE or chronic wasting disease - CWD). These disorders are characterised by spongiform changes within the neural tissue resulting in neuronal loss and this occurs without an inflammatory reaction. Furthermore, the incubation period is typically very long. The main pathological event that is thought to occur in these diseases is the conversion of a benign cellular protein (cellular prion protein, PrPC) into a unique conformation called PrPSc. This misfolded protein then accumulates in neuronal tissue, a process accompanied by neurodegeneration. TSE transmission is a considerable concern after the interspecies transmission of BSE to humans and other animal species via presence of cattle BSE in meat/feed. To date, many therapeutic approaches have been tested in vitro and in vivo, targeting the cellular PrP, misfolded PrPSc or even the whole process of PrPC misfolding. It has been established that heterologous or homologous recombinant prion protein can inhibit TSE disease progression in vitro and in vivo. The proposed project will look for development of the recombinant prion protein with the main focus on ovine prion protein and its polymorphic positions (136, 154 and 171) as a model. The potential therapeutics will be tested in the 'protein misfolding cyclic amplification' (PMCA) which is a proven method for the in vitro propagation of misfolded prion protein. Potential therapeutic candidates will be also tested with range of prion diseases and in infected with prions cells.
Organisations
People |
ORCID iD |
| Kevin Gough (Primary Supervisor) | |
| Jeremy Roberts (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M008770/1 | 01/10/2015 | 31/10/2024 | |||
| 1804229 | Studentship | BB/M008770/1 | 01/10/2016 | 20/01/2021 |
| Description | Previously, ovine recombinant prion proteins from protein natural variants were produced and shown to inhibit the in vitro incorrect folding of prion protein. In ovine scrapie three polymorphic position in prion protein sequence determine the disease susceptibility or resistance (ovine positions 136, 154 and 171). Here, rPrP variants were produced based on genotype background. The most effective inhibitors were V136R (valine replaced by arginine), V136L (valine replaced by leucine), V136K (valine replaced by lysine) and V136P valine replaced by proline) mutants. These new inhibitors were characterised by dose-response experiments and all had IC50 values <10 nM and were between 12 and 57 times more potent than natural variant rVRQ. rRRQ, rPRQ and rKRQ were further characterised and shown to inhibit a range of ruminant prion PMCA reactions including with distinct prion strain seeds (scrapie isolates, bovine BSE, ovine BSE). Moreover, the rRRQ was also shown to effectively protect Rov9 cells from scrapie infection when applied at 250 nM. The study demonstrated for the first time that the rPrP sequence can be mutated at sites known to be involved in prion disease susceptibility, to produce inhibitors with improved efficacy. |
| Exploitation Route | The results of this research could be expanded into testing new inhibitors using different protein misfolding methods. Similar approach could also be used to investigate other polymorphic positions in ovine PrP and investigate their effects on protein misfolding inhibition. More importantly, research in this filed could focus on further characterisation of rPrP mutants for other species, for instance position 129 in human rPrP is polymorphic. Research focusing on investigating recombinant human PrP in the inhibition of in vitro prion protein misfolding could be designed with focus on position 129 and also other amino acids substitutions could be implemented in human PrP at position 129. |
| Sectors | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Category 3 Laboratory policies and practice |
| Geographic Reach | Local/Municipal/Regional |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Impact | As a member of Category 3 Laboratory team I am involved in creation and improvement of policies and procedures required by everyone working in the Cat 3 Laboratory environment. All the precautions are taken because of the risk correlated with work with prion diseases. Every described procedure and operation must reduce the risk of contamination of the laboratory environment and the staff or students working in this area. |
| Description | Postgraduate Training - Post-genomics and Bioinformatics |
| Geographic Reach | Multiple continents/international |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Description | Microbiology Society Conference Grant 2017 |
| Amount | £270 (GBP) |
| Organisation | Microbiology Society |
| Sector | Learned Society |
| Country | United Kingdom |
| Start | 02/2017 |
| End | 04/2017 |
| Title | Making recombinant prion proteins with mutated position |
| Description | As a part of my project I used site-directed mutagenesis to change one amino acid position in ovine prion protein sequence (PrP). This position was mutated into 9 different amino acids and each of newly created recombinant PrP was purified by myself. This have lead to creation new recombinant prion protein, which are currently being used in my research. |
| Type Of Material | Biological samples |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | At this stage, the newly created recombinant proteins are being tested in various reactions, therefore the impact is still being investigated. |
| Title | Rov9 cell line stably infected with ovine scrapie |
| Description | During this study a cell line (Rov9) infected with infectious scrapie prions was created. This cell culture model of scrapie infection was used to validate previous results of the inhibition of prion protein misfolding. Infection optimisation steps included testing couple scrapie field and experimental isolates and potential use of processed or not processed brain homogenates or purified PrPSc. |
| Type Of Material | Cell line |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | The produced in this research scrapie infected cell line will be very beneficial to research group. The scrapie model allowed to test potential inhibitors of prion protein amplification in cell culture system. The study presented here is based on a model TSE, scrapie in sheep but similar approach could be used for other species including human, where therapeutic intervention is highly required for human TSE diseases. The ovine rPrP mutants could be tested against models of human TSEs to determine their direct therapeutic potential and an analogous strategy for mutating and testing human rPrPs could be followed. |
| Title | Publication Vilette et al. Title: Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein |
| Description | This work by Vilette et al., describes the creation and use of rabbit Rov9 cells that were engineered into producing ovine cellular prion protein |
| Type Of Material | Database/Collection of data |
| Provided To Others? | Yes |
| Impact | This work allowed to use the cell line and gave significant knowledge to create infected with scrapie prions cell line during the current study |
| Description | Activity for kids on the SB Fest |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | In 2007 I was part of the group preparing SB Fest scientific part. We were involved in making the lesson about blood. It included presentations about blood and blood models. The activity was directed to school pupils (up to 12 years old) and the purpose of the activity was to teach young people what is blood, what components it has. At the end of the activity, children were asked to complete a short world search puzzle. By the end of the presentation, the audience was familiarised with blood, its components and their main functions. |
| Year(s) Of Engagement Activity | 2017 |