Understanding the role of DNA repair in Huntington's Disease pathogenesis: towards new therapeutic targets

Background and aims:

Huntington's disease (HD) is an inherited degenerative brain condition in which patients develop a mixture of symptoms including involuntary movements, changes in mood and behaviour, and dementia. The disease progresses slowly but relentlessly over 15-20 years, and is life-shortening. We have no treatments that can prevent or slow it. Patients often have complex care needs over a long period of time and these put a considerable strain on their carers and families, as well as on healthcare resources in general. Although HD is a rare condition (affecting about 1 in 8000 people), it is one of a family of over 30 diseases caused by expansion of repeating sections of DNA in genes. Together these conditions are estimated to affect over 3 million people worldwide (1 in 2000) at considerable human and economic cost.

The genetic mutation causing HD was identified in 1993 but it is still unclear exactly how this leads to specific nerve damage and loss in the brain. The mutation consists of expansion of a repeating 'CAG' sequence in the DNA of the huntingtin gene. Unaffected people have between 9 and 35 CAG repeats; HD patients have at least 36, and generally the greater the number of repeats, the earlier the disease starts. The brain nerve cells that are most affected by the disease harbour more repeats than other cells in the same patient.

A recent genetic study involving our group and others identified various DNA repair factors as determinants of the age at which HD symptoms start. We think that DNA repair processes might directly cause an increased number (expansion) of CAG repeats in vulnerable cells and lead to cell death and the onset of disease. The aims of this project are to characterise how this process occurs, and to try to identify ways in which it could be blocked.

How the project will be carried out:

It is difficult to study cellular processes in the brains of living HD patients and so we will use a variety of experimental methods to test our ideas.

1. Genetics. We will look for variants in the DNA repair genes of patients who have developed HD at an unexpectedly early or late age compared to that predicted from their CAG repeat length. We expect that this will identify new variants that will give insight into how and when HD starts.
2. Cell culture. We will develop a model system for testing CAG expansion in nerve cells grown in the laboratory. Once this system is established we will test the effects of the DNA repair variants on CAG expansion. Factors that lead to greatly enhanced repeat expansion could represent novel therapeutic targets: if we can block this process we could theoretically delay disease onset.
3. Biochemistry. To work out exactly how DNA repair proteins interact with CAG repeats in DNA and cause them to expand, we will purify the different components and analyse their reactions in the laboratory. We will be able to plug the most interesting mutations from our genetic screen into the biochemical analysis to uncover more detail about mechanism.
4. Inhibitors of repeat expansion. We will test compounds that can block DNA repair proteins in our CAG expansion system. If they can block CAG expansion then they could represent new therapeutic leads with the potential to alter disease onset.

Therefore this project will add important insight as to how the HD mutation leads to disease, linking genetics with cellular and molecular biology. It could elucidate mechanisms of repeat expansion and identify new drug targets, giving a clear link from bench to bedside. These findings are likely to be beneficial in other DNA repeat diseases as well as HD given that the underlying pathogenic mechanisms are probably similar. More broadly, insights into the cellular mechanisms underlying HD may well reveal novel aspects of neuronal biology that are useful in the investigation of other types of neurological disease and dementia.

Huntington's Disease (HD) is a life-shortening, progressive, autosomal dominant neurodegeneration characterised by chorea, psychiatric disturbance, and dementia. Although it is rare (1 in 8000), patients often have long-term care needs that lead to considerable emotional and financial strain on families, carers, and healthcare systems. HD is caused by a CAG repeat expansion in the HTT gene that results initially in striatal medium spiny neuronal degeneration in the brain. There are currently no disease-modifying treatments. Previous work has implicated DNA repair processes in triggering CAG repeat expansion in striatal neurons, thereby accelerating the onset of HD. The work proposed here aims to address two questions: 1. Are DNA repair proteins (in particular FAN1) and their variants directly involved in CAG repeat expansion? 2. Can selective inhibition of DNA repair pathways slow or prevent repeat expansion?

Genetic, cellular and biochemical approaches will be used to test these hypotheses. Novel variants in DNA repair genes associated with especially early or late disease onset will be identified through exome sequencing of a stratified HD population. A robust system for inducing and measuring CAG repeat expansion and cell viability in model HD cell lines (mouse and human) will be established, and then the effects of repair gene variants analysed. Certain DNA repair proteins of interest (including FAN1) will be expressed and purified, and their activities on CAG repeat-containing DNA assayed in vitro. We will then investigate whether small molecule inhibitors of DNA repair enzymes can abrogate CAG repeat expansion (and hence improve neuronal viability) in our cellular and biochemical assays. If they can, future exploitation of these leads could ultimately yield new drugs with the potential to delay or prevent disease onset. This would represent considerable progress in HD therapy and likely be applicable to the wider family of trinucleotide repeat disorders.

Rare diseases (of which there are over 7000, including HD) collectively affect approximately 1 in 17 people (i.e. 3 million in the UK, 350 million worldwide). Recognition of this health burden and the fact that understanding of rare diseases has often led to insights into common conditions has led to prioritisation of rare diseases research (UK strategy 2013; www.europlanproject.eu). Since more than 80% of rare diseases are thought to have a genetic basis HD is an excellent model rare disease for investigation.

This project will have an impact on various groups. Some will benefit immediately (e.g. academics- described elsewhere) whereas others will gain in the medium to long term:

1. Individuals and families affected by HD
This project will use genetics and molecular biology to understand how and when CAG repeats cause HD. Individuals carrying the HD mutation and their families will gain greater understanding of the condition, empowering them and helping them make appropriate arrangements for their home lives in a timely fashion. This project may enable clinicians to begin to stratify patients by genetic modifier risk and hence give patients information about disease onset and development: a first step towards truly personalised Medicine. If new drug targets are uncovered, novel therapeutics could be developed in the longer term with clear impact.

2. Healthcare professionals
If we understand HD pathogenesis and its modifiers in more detail clinicians (and others) can better explain a complex condition to patients. We can also begin to stratify patients by predicted age at onset or phenotype (e.g. motor, behavioural, or psychiatric) and hence offer better care for each individual. As well as being good for patients, this would improve targeting of resources and be of economic benefit.

3. Researchers
The project will enable the training of junior researchers (e.g. we have recently been awarded a competitive PhD studentship) to help develop the next generation of biomedical research leaders. It will also benefit me as I develop a research portfolio and profile in academic Neurology, improving my prospects of further fellowships and grants as I move towards research independence.

5. Development of Therapeutics
If we show that DNA repair enzymes are involved in somatic CAG expansion we will test inhibitors of the process as a first step towards therapeutic development (collaboration with Prof Jackson (Cambridge)). Although the development of therapeutics is beyond the scope and timescale of this project, there is the potential for significant impact in this area given the lack of any disease-modifying drugs in HD and the increased interest and profitability in orphan drugs (http://www.orpha.net). I envisage further grant applications relating to this in due course. This potential impact dovetails with the one of the goals of the International Rare Diseases Research Consortium (IRDiRC): 200 new therapies for rare diseases by 2020 (http://www.irdirc.org/goals).

6. Wider society
HD imposes a high health and economic burden on society due to patients having wide-ranging care needs for a long period of time (often >15 years). Advances in understanding and treatment could reduce these costs significantly. Positive results in this project would raise the public profile of HD and lead to increased funding through charities (e.g. HDA, EHDN). Results will also be broadly applicable to neurodegeneration and dementia: particularly relevant in today's ageing population. Policy makers in the EU have acknowledged that these areas have unmet research needs and set up the Joint Programme of Neurodegenerative Disease Research (JPND; http://www.neurodegenerationresearch.eu) under the umbrella of Horizon 2020 research funding. The JPND is the largest global neurodegeneration research initiative, and so if this project is successful I envisage the ability to leverage significant new funding for further investigations.