Developing cell lines that propagate human prions

Prions are misfolded forms of a normal protein that cause a rapidly progressive, incurable dementia. These often present with problems with memory, language, balance, and co-ordination, and are always fatal; often in just a few months. They can occur in animals such as sheep, goats and cattle, and in humans - where they cause conditions like sporadic Creutzfeldt-Jakob disease (CJD) and variant CJD, which remains a major public health concern owing to its transmission to humans from cattle infected with bovine spongiform encephalopathy (BSE). The lifetime risk of someone developing CJD is 1 in 5000, and there are no treatments currently available that can change the course of the disease.

The misfolding of other normal proteins to cause disease, in a similar fashion to prions, is a concept that is also becoming increasingly important in understanding other, more common degenerative brain conditions like Alzheimer's disease. Indeed, many recent advances in understanding these conditions have sprung from research on prions that cause CJD. Studying prions closely could therefore shed light on other devastating conditions that cause dementia.

Although we know that prions cause disease, we still don't know how they spread, exactly how they damage the brain or have any effective treatments for them. To answer these questions, we need to be able to study human prions closely in a stable environment. This would also allow us to directly measure the number of prions in tissue, and could act as an important marker of disease progression or response to new treatments.

Growing cells outside of the body (cell culture) and infecting them with prions is a good opportunity to do this, but it has proved difficult to find the right cells to allow this. A group from the Medical Research Council (MRC) Prion Unit has previously developed cells that can be infected with mouse prions. This technology has enabled huge advances in understanding how these prions spread and damage cells, and shown that this approach is promising.

However, no one has grown cells that can be infected with human prions - which is crucial to better understand these conditions and test potential treatments we could use to help patients. In this proposal we aim to develop a human cell culture model of prion infection. Work will be carried out at the MRC Prion Unit, a world-leading centre of prion biology. The Unit works very closely with and shares senior staff at the National Prion Clinic, a national referral centre for patients with prion disease, and organiser of the largest single-centre clinical trial in prion disease and prospective Cohort study of human prion disease.

Using the extensive expertise of Unit staff, we propose to utilise an experimental "reconstitution" approach previously used by Prion Unit staff to successfully develop cells able to maintain a mouse prion infection: by silencing the expression of the normal prion protein of mouse cells, and inducing it to express human prion protein. Previous work at the Prion Unit and internationally with animal models has shown that this approach should render these mouse cells capable of maintaining a human prion infection, once the optimal conditions for cell growth are identified. The Prion Unit would then be ideally placed to develop and disseminate an automated process that would revolutionise the field of human prion research internationally, as it successfully accomplished with mouse prions.

Prion diseases share a mechanism related to seeded polymerisation and aggregation of a misfolded form of the normal prion protein. Other more common neurodegenerative conditions, including Alzheimer's disease, are also associated with misfolded protein aggregates; a wealth of evidence has led the field to pursue so-called "prion-like" mechanisms as fundamental to the entire group.

Animal models have traditionally formed the basis for research into prion diseases, but are cost, time and resource-intensive.

Cell lines are invaluable for in vitro studies of many complex diseases processes. Mammalian cell lines that propagate mouse prions have significantly advanced prion biology, and the development of an automated mouse prion infectivity assay at the Medical Research Council Prion Unit has revolutionised their study. However, there are no cell lines able to reproducibly propagate human prions - this remains one of the crucial goals of research into prion diseases.

The primary objectives of this proposal are:
1. Develop cell lines that can reproducibly propagate human prions
2. Establish an automated system for rapid and accurate titration of human prions

We propose using a "reconstitution" approach, analogous to successfully established prion animal models. Short hairpin RNAs and negative live cell sorting will be used to silence native prion protein expression in cells susceptible to prion infection, followed by reconstitution with human prion protein, infection with human prions and sequential single cell cloning.

Established lines will be used to develop an automated human prion infectivity assay, permitting direct titration of prions in body tissues (analogous to HIV viral load), as a diagnostic and therapeutic biomarker, and allow the crucial direct study of human prions in vitro. Understanding the fundamental mechanism of how prions propagate and cause neuronal damage is likely to have wider significance for common neurodegenerative diseases.

Research community and industry impact

The successful propagation of human prions in vitro is rightly regarded as one of the most crucial research goals in prion biology. By developing this technique and widely disseminating it through academic publications and presentations, we expect the research and industry communities to significantly benefit.

By effectively quantifying human prion titre in tissue, many of the crucial unsolved problems regarding prions would become immediately tractable, including: a rapid method for differentiating and further studying the different prion strains, which are fundamental to their widely variable clinical phenotypes and prognosis; a potential disease and therapeutic biomarker, analogous to an HIV viral load. The latter is of particular importance as many international groups are developing therapeutics that interfere with prion propagation, and have no way to directly assess biochemical response at present - this severely impacts and delays the development of clinical trials. This includes the future clinical development through trials of PRN-100, a fully-humanised monoclonal antibody therapy developed by the MRC Prion Unit and D-Gen Ltd, with support from the Medical Research Council.

The MRC Prion Unit has cultivated collaboration with industry in the search for an effective therapeutic agent for human prion disease. An ongoing research programme supported by GlaxoSmithKline is screening small molecule compounds for anti-prion efficacy; the most advanced compound is currently in studies aiming to develop a phase I ready drug. However, such approaches within the Unit and internationally rely upon cells infected with mouse prions, a proxy system that inadequately mimics the human infection and would be immediately improved were this project to be successful.

Policy and Patient Impact

Human prion diseases, particularly variant CJD, represent a major ongoing public health concern. Whilst the vCJD epidemic peaked in incidence in 2000, and since then has been in decline, it is well-known that human prion disease incubation times can extend over 50 years. One in 2000 healthy individuals is assumed for public health purposes to be infected with vCJD, posing risks for onwards transmission. Until this year, all reported definite vCJD patients had a methionine homozygous genotype at polymorphic codon 129 of the prion protein gene, shared with 40% of the healthy population. We have now diagnosed at autopsy the first patient with the most common methionine-valine heterozygous genotype, potentially marking the start of a "second wave" of vCJD. For these reasons, it is likely that vCJD will remain on the policy agenda of government for some time.

Policy-makers and the Department of Health are required to address these problems by issuing guidance on prion decontamination and infection risk. The MRC Prion Unit has played a leading role in advising successive governments on potential health risks posed. Unfortunately, current guidance is based largely on small animal studies, with little or no prion titre data; hundreds of millions of pounds are spent annually on prion decontamination and infection control, which may be inappropriately allocated. This project will develop a system to propagate human prions in vitro and allow direct quantification of prion titre in various biofluids. This will provide a tool to allow for experiments to develop direct evidence about measures aimed at preserving the Public Health and therefore to inform government policy. Furthermore, accurate knowledge of infective prion content in various body compartments would enable a considerably more accurate risk assessment for exposed patients (for example, through surgical procedures), and help to improve much of the psychological morbidity involved with uncertainty over their exact risk.