Novel therapeutic strategies to target RAN translation of pathological C9ORF72 repeat transcripts and associated neurodegeneration

The basic units of life, cells, convert food and oxygen into energy to produce proteins, constituents indispensable to the building and functioning of cells. The diverse roles of proteins and their levels account for health or diseases. The blueprint providing the information for making up thousands of proteins is housed under the form of genes in the cell centre (nucleus). Small messenger molecules copied from the blueprint are transported out of the nucleus where they serve as individual instruction manuals for manufacturing each protein within specialized factories called ribosomes.

Extensions of a short repeated motif in a gene called C9ORF72 are the most common changes causing two incurable and lethal neurodegenerative diseases in adults, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Approximately 10,000 individuals suffer from ALS and FTD at any time in the United Kingdom. Progressive injury and death of nerve cells in the front and side parts of the brain provokes characteristic changes in the personality and behaviour of patients suffering from FTD. On the other hand, the progressive paralysis observed in ALS is due to the death of motor nerves that control movements by transmitting signals from the brain and spinal cord to the muscles. A proportion of patients with ALS disease also develop FTD, and conversely, many patients with FTD will eventually develop ALS. Whilst no treatment is available for FTD, the current standard of care for ALS patients, riluzole, only marginally extends survival. The identification of therapeutic targets to cure or slow down the progression of these neurodegenerative diseases is therefore of paramount importance.

A prominent mechanism by which large extensions of the C9ORF72 repeated elements are thought to cause death of nerve cells involves the production of aberrant C9ORF72 messengers guiding the building of toxic proteins that provoke multiple cellular damage mechanisms. Although riluzole might moderately increase the survival of patients with C9ORF72 related ALS, it does not provide a cure and does not prevent the production of abnormal biomolecules.

We recently identified some of the specialized molecules serving in the factories where the C9ORF72-repeated proteins are produced. We also found that reducing their number can halt the manufacture of the toxic proteins and stops cells from dying. These findings were made in a human kidney cell model routinely used in laboratories around the world. We now want to test whether this groundbreaking discovery holds true in disease-relevant models of C9ORF72-ALS/FTD using a fruit fly animal model and human nerve cells made with cutting-edge scientific technologies from the skin of C9ORF72-ALS/FTD patients. In this project, we also want to study the global effects that reducing the levels of the specialized molecules may have on the production of the other normal proteins and test whether they are directly involved in the manufacture of the abnormal repeated proteins.

In summary, this research will test a novel concept for a potential treatment of patients suffering with C9ORF72-linked ALS/FTD in the near future. This novel strategy could also potentially be useful for other diseases including Fragile X-associated Tremor/Ataxia syndrome, Alzheimer's disease and some forms of cancer. Several collaborating communities of researchers including scientists and clinicians will be involved with the research. Furthermore, this state-of-the-art science will lead to inspirational communications, presentations and publications that will benefit the academic community, the general public, local schools, the tertiary sector and potentially in the longer term, healthcare professionals and the pharmaceutical industry.

Hexanucleotide repeat expansions in the C9ORF72 gene are the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of fatal adult-onset neurodegenerative diseases. FTD involves neuronal loss in the frontal and temporal lobes of the cerebral cortex, leading to cognitive and personality alterations. ALS is characterised by selective degeneration of upper and lower motor neurons, progressive paralysis and death usually within 3-5 years from symptom onset.

The pathophysiology is complex, potentially involving haploinsufficiency, sequestration of RNA-binding proteins and production of dipeptide-repeat proteins (DPRs) by repeat-associated non-AUG (RAN) translation. Recent evidences, including my own research, point DPRs as a primary driver of pathogenesis in cell and animal models of C9ORF72-ALS/FTD.

The mechanisms driving RAN translation of C9ORF72-related DPRs are unknown. We recently uncovered two RAN-associated translation factors using transfected human embryonic kidney reporter cell models. We will now test whether partial and single depletion of these RAN translation factors inhibits DPR production in neurons derived from C9ORF72-ALS/FTD patients and in a Drosophila model of disease, and respectively prevent neuronal death and neurodegeneration-associated motor deficits. We will also investigate the C9ORF72 repeat RNA-binding activities of the RAN translation factors using RNA immunoprecipitation experiments in cell models and in vitro biochemical assays with recombinant proteins. The potential co-localisation of the RAN translation factors with C9ORF72 repeat transcripts will also be assessed in vivo by dual immunofluorescence and fluorescence in situ hybridisation studies in human C9ORF72-ALS/FTD brains. Finally, transcriptomics and polysome profiling assays in human cells depleted of these factors will allow investigation of genome-wide effects on the expression and translation of cellular transcripts.

The primary beneficiaries from this research will initially involve members of the academic sector, particularly scientists and clinicians working on the expression of genes and neurodegenerative diseases as described in the academic beneficiaries section. During the course of the project, this research will also have an impact on patients suffering from motor neuron disease, their carers and families as well as on the staff and students in our institutions. Research outcomes in the longer term will potentially be of interest to additional beneficiaries from the public sector such as healthcare professionals and the private industry sector for the development of therapeutics.

The general knowledge from this research project will also inform public and tertiary sectors on future research development and priorities in the field of age-related neurodegeneration and neurodegenerative diseases. These include the research councils (mainly MRC and BBSRC), the professional associations (British Neuroscience Association, Association of British Neurologists, Age UK) and charitable trusts (Motor Neurone Disease Association, Sheffield Motor Neurone Disorders Research Advisory Group, ALS worldwide). Local schools and the public will benefit from general concepts concerning the brain, nerve cells, the expression of genes and the dysregulation of these processes during ageing of the brain and neurodegeneration. They will also be informed about the development of the research through sharing interesting findings as well as hearing about research challenges.

My research aims to provide novel therapeutic targets to treat C9ORF72-linked amyotrophic lateral sclerosis and frontotemporal dementia. There is currently no cure for these lethal neurodegenerative disorders that have devastating impact, socially, emotionally and economically, upon the patients, their families, their carers and the NHS. Due to the adult-onset nature of these diseases, the number of cases is expected to grow in an ageing world population. Findings from this research may also be directly applicable to other fatal and incurable microsatellite expansion disorders including Huntington's disease, spinocerebellar ataxias, myotonic dystrophy, Friedreich's ataxia, Fragile X-associated Tremor/Ataxia Syndrome and some forms of cancer.

The proposed research is also expected to lead to the development of commercial opportunities. The PI is the primary inventor of a patent application related to this proposal for the use of dipeptide repeat protein antagonists in the treatment of neurodegenerative disorders by gene therapy approaches (PCT/GB2017/051539). Any other commercially sensitive findings resulting from the analysis of our profiling studies for example will be handled confidentially and appropriately with our University Research and Innovation Services and their approved partners.