New humanised mouse models for dissecting the pathobiology of disease, using FUS-ALS as a paradigm

Humans and mice are descended from a common ancestor and so we share genes and have similar diseases caused by similar mutations in our equivalent genes (known as orthologs). Therefore to understand more about human disease we work with 'mouse models' that have the same mutations and manifest similar symptoms.

Many neurodegenerative diseases run in families as 'dominant' diseases, so if your parent has the disorder there is a 50% chance you will inherit the mutant gene, and also succumb to the disease. These dominant disorders are modelled by mice called transgenics, which carry human mutant genes and so get the disease in a similar way to humans.

Transgenic animals are made by incorporating the human mutant 'transgene' into the mouse DNA. Unfortunately when this happens the human mutant gene is usually incorporated in lots of copies, somewhere fairly random, not just a single copy as it would be normally in humans. One result is the mouse produces considerably more than the usual 'physiological' amount of mutant protein, and more often than not the mouse develops symptoms that simply come from having too much protein, rather than the actual effects of the mutation. This means the mouse is a far less accurate model than it could be.

Also, sometimes the human and mouse biochemistry of the mutant protein can be different, so simply making the same change in the mouse gene does not produce an accurate disease model.

We have developed a new type of mouse model in which we replace the mouse gene with the complete human gene, both the normal human gene and the mutated human gene in different mouse strains. Therefore we can look at what happens to the mouse when the human genetic material is expressed at normal levels, and this will give us new insight into disease processes. Our new mouse models may be a Refinement of existing models.

As an example, we have chosen to work with a gene called FUS, which is mutated in an untreatable neurodegenerative disease called amyotrophic lateral sclerosis (ALS, also known as motor neuron disease). ALS is an incurable and relentless devastating neurodegenerative disorder which causes progressive loss of muscle function and paralysis. ALS leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of ~1 in 300 by 85 years old, in UK. ALS is generally a mid-life disease affecting people in their 40s and 50s although there is a wide age-range of symptom onset and aggressive FUS-caused ALS has been described in children as young as 11 years old. FUS is also occasionally mutated in other incurable neurological diseases.

Now we are applying for a three year grant to:

(1) extend our method to make a slightly different type of mouse called a conditional mutant, which allows us to turn the mutant protein on in different tissues at different times. This will help us work out what goes wrong in ALS and other disorders. And,
(2) to fully characterise our new FUS mouse mutants, so we can learn more about the way that mutant FUS causes nerve cells to die. If we know this, we are step further along to therapies for ALS and other neurodegenerative diseases.

Our technology can be used for any gene, not just FUS and for example, we are well along with using this method for SOD1, which is another 'ALS gene'. So we have developed a new technology that we believe will be widely used by other laboratories, and we want to characterise the mice we have already created to understand more of how neurodegeneration happens, so that we can better understand human disease and how to treat it.

Many genes, including the well-known genes FUS, TDP43, SOD1 that are causative for the neurodegenerative disease amyotrophic lateral sclerosis (ALS) are dosage-sensitive.

ALS is an incurable and essentially untreatable disease that leads to paralysis and death through the progressive loss of motor neurons. ALS is generally a mid-life disorder but patients as young as 11 years of age (with FUS mutations) have been described. Most familial forms of ALS arise from dominant mutations and are modelled in mice by overexpression of the human mutant transgene. However, phenotypes arise in transgenic mice from overexpression per se, rather than from the effects of the mutation.

To create more sophisticated models that overcome issues of dosage-sensitivity and express the biochemically correct human protein at physiological levels, we have developed a new technology that is straight-forward and should be useable by standard-gene targeting laboratories.

We have created 'genomically humanised' animals in which the mouse gene is entirely replaced by the human genomic locus. These mice express the mutant protein at physiological levels. We are now applying for funding to achieve two goals,

(1) to develop our genomic humanisation technology to create conditional mutants and,
(2) to characterise our new mouse lines and shed light on the biology and pathology of our paradigm, FUS, in health and in neurodegeneration.

By the end of this program of research we will have informative new FUS models for the community, and a better understanding of how mutant FUS causes neurons to die. The normal biology of FUS is not well understood and FUS mutations can be particularly aggressive, often causing ALS in young teenagers and adults, for as yet entirely unknown reasons. FUS is also involved in other neurological diseases, and is an important target for our attention.

This technology is applicable to any organism for which ES cells exist, not just mice.

The impact of this application lies in the fields of neurodegenerative/neurological disease, and in mouse modelling, and the beneficiaries are potentially commercial Pharma and Biotech, patient and carer groups, academics. The work described here could contribute to health and wealth outcomes, and have an impact on biomedicine contributing to quality of life outcomes.

With respect to the mouse modelling aspects of this application, development of this technology will enable all laboratories that carry out gene targeting to humanise mouse models, as the basis of our approach is classical gene-targetting and therefore this project may have significant impact both commercially and academically, in providing more tailored models and so more accurate models for human disease. This has both health and wealth, and quality of life impact.

Further, this technology is applicable to all organisms for which ES cells capable of homologous recombination exist, the number of such organisms is increasing dramatically and includes both rat and human. In human we can see uses in gene therapy in correcting relatively large regions of the genome, for example in iPS cells.

With respect to neurodegenerative disease/neurological disease, we are specifically interested in analysing FUS humanised mouse mutants, and this project came about because of the lack of FUS models due to the dosage sensitivity of FUS in such animals -- and similar difficulties in other models (e.g. with the TDP43 gene), being created to model human neurodegenerative disease. Thus we have invested in developing a new technology to create animals with the correct human protein biochemistry arising from the mutant human gene, and in expressing the gene at endogenous levels. We see this as essential for understanding not only how mutant FUS causes disease, but how other dosage-sensitive genes, in ALS and all other areas of biomedicine, give rise to pathology.

Thus the impact of our specific biological investigation lies in dissection mechanisms of FUS-ALS and neurodegeneration, and in a better understanding of the role of disruption of RNA metabolism in human disease. The beneficiaries are commercial organisations investigating such diseases - and we note our excellent ties to Pharma and Biotech, through current collaborations and through the extensive opportunites for networking and development provided by UCL and UCLBusiness. The beneficiaries are also patient organisations ultimately as treatments and cures are eventually found for ALS (and other forms of neurodegeneration). We also note that new - commercial and academic -- approaches to treating ALS entail reduction of levels of transcripts from mutant alleles and our mouse models offer the only genomically accurate models for developing such therapies.

We also note we are excellently placed to follow up findings with translational neuroscience, as the adjacent National Hospital for Neurology and Neurosurgery hosts one of the largest ALS clinics in Europe and clinicians at that clinic are joint appointed to our Departments as clinician-scientists working on projects related to this application.

With respect to training, we have a range of students at all levels working in our labs including undergraduate neuroscientists and geneticists from UCL, UK and abroad, Master's students from UCL and elsewhere in UK, PhD students, MD students, and a range of clinician scientists. Thus we provide an unusual and important training in the creation, validation and characterisation of novel engineered mouse models of neurodegeneration, for new generations of scientists and clinicians.

We hope that the new models we are developing will Refine the use of mice as disease models, in accordance with the 3Rs.