Research done at SITraN

Current treatments and condition management for MND

Many clinical trials have been undertaken around the world to test new therapies with the potential to slow down the progression of motor neurone injury. Of these only riluzole has been shown to have a definite effect in slowing the progression of MND. The overall effect of riluzole is quite modest – though some patients may have a really good response. However, we clearly need to build on this effect and it is likely that several medications may be required to protect the motor neurones in a more robust way.

Other treatments are aimed at alleviating symptoms and maximising the motor ability of MND patients. The clinical care teams need to pay particular attention to overcoming the problems that may arise when breathing and swallowing functions become impaired.

SITraN: New hope for MND

2010 sees the opening of the Sheffield Institute for Translational Neuroscience (SITraN).

Over the last four years Professor Pam Shaw and her team from the University of Sheffield, in partnership with the Patrons of the Sheffield Institute Foundation Charitable Trust, have raised £16m for the creation of the Sheffield Institute of Translational Neuroscience (SITraN) together with key new academic positions. SITraN is a new 2,800m2 research facility within the central campus of the University of Sheffield. It will house 150 researchers, providing state of the art laboratory facilities, with the mission to advance understanding of the mechanisms of motor system neurodegenerative disease and translate this knowledge into neuroprotective therapies resulting in improved outcomes for patients. 

So what will happen at SITraN?

SITraN will allow our programmes of research to be developed and extended – integrating clinical and laboratory studies, aimed at identifying what leads to the degeneration of neurones at the root of MND. The creation of SITraN will allow us to double our research capacity and bring in new scientific skills to our interdisciplinary team. 

What is Translational Neuroscience?

Translational Neuroscience means that scientific developments emerging from experimental work in the laboratory will be translated into effective therapies for patients in the clinic.

Research at this Institute will also include work on other degenerative conditions such as Parkinson's disease, Spinal Muscular Atrophy (SMA - a childhood form of MND) and Alzheimer’s disease as they all have some common ground. Discoveries leading to neuroprotection in one of these disorders could have an important treatment impact for the others.

The SITraN MND research team is in a unique position to make a major contribution to the search for the causes and curative treatments for MND. Research resources underpinning the programme include:

An extensive clinical database of MND patients with high quality clinical details of all patients.
A large resource of brain and spinal cord material (donated by 165 MND patients), as well as DNA and cerebrospinal fluid banks.
Robust cellular and other in vivo experimental models of MND in which new treatment approaches can be verified.
The research team assembled has multidisciplinary skills in neurology, pathology, gene therapy, molecular genetics, protein chemistry, cell biology and pharmacology to facilitate identification of the mechanisms involved in motor neurone degeneration and targets for therapies by which to protect the motor neurones from injury.

Laboratory programmes

Patients with MND Parkinson’s disease and SMA urgently need therapies that slow down, or even halt, the progression of their diseases.

Within SITraN we will exploit recent advances in MND and PD genetics to build an effective pipeline of research that can:

1 Identify which pathways, and which molecules (as candidate drug targets) within the pathways, are crucial for the processes of degeneration in the nervous system. For this we will use a range of experimental model systems and gene expression profiling which allows us to understand the complex cell biochemistry that goes wrong to cause injury to nerve cells

2 Identify drug-like molecules from large ‘libraries’ of compounds that have the correct activity to modify the disease causing biochemical pathways

3 Screen the most promising neuroprotective agents in a systematic cascade of disease models to identify those that should be followed up and developed as potential human therapies

4 Develop new ways of getting treatments into the brain across the blood brain barrier. For this we will use tiny polymer ‘nanoparticles’ and gene therapy using safe viral vector technologies.

 

We will use our scientific skills to investigate the role of motor neurone neighbourhood cells (glial cells) and how these cells contribute to motor neurone injury and the spread of the disease. We will also investigate the transport system within motor neurones and what goes wrong with this to cause a disconnection between the nerve terminal and the muscle.

Exciting recent scientific developments now allow skin cells taken from living people to be converted into stem cells and then motor neurones or glial cells. This will enable us to extend our model systems by which to understand human neurodegenerative conditions and test out new treatment approaches. 

Examples of the clinical programmes
Translating insights from scientific research in the lab into clinical trials is now much more possible with the founding of the Institute.

1 Therapeutic trials of novel neuroprotective agents in MND 
Professor Shaw and her team play a key role 
in multiple national, European and international trials of potential neuroprotective agents, but also play a role as champions for patients, by negotiating with NICE and Health Care Commissioners when new treatment approaches, such as riluzole and non-invasive ventilation, become available.

2 Supporting respiratory function in patients with MND 
This is particularly important as neuromuscular respiratory failure is the usual cause of death in patients with MND. We showed that non-invasive ventilation had a major positive impact in extending life and improving the quality of life and this work has recently 
resulted in the publication of a NICE guideline for the management of this problem. Thus the Sheffield group, with collaborators in Newcastle, have been responsible for demonstrating the therapy with the greatest positive impact on survival so far demonstrated in MND.

In ongoing work we are investigating how to improve the treatment of patients with weakness of the respiratory muscles. We are investigating whether a cough assist device is helpful in the prevention of chest infections and whether stimulating the diaphragm breathing muscle with a pacemaker may be helpful in easing respiratory symptoms.

3 Identification of new genetic and environmental predisposing factors for familial and sporadic motor neurone disease (MND).

Raising Funds for an important piece of equipment which will help finding the gene which is responsible for MND. The Illumina HiScan system.

 

The research group within the Sheffield Institute of Translational Neuroscience (SITraN) study neurodegenerative diseases such as motor neuron disease (MND) and Parkinson’s disease (PD) in order to understand the fundamental causes of these conditions and devise new ways of protecting the nerve cells from injury. We are interested in investigating at a molecular level, the genetic and epigenetic mechanisms which may be determining the development of particular neurodegenerative conditions.

Whilst investigations have been carried out previously and uncovered a number of genes that are responsible for the familial disease, it remains unclear what triggers the disease in sporadic cases. A thorough investigation of genetic and epigenetic effects requires the examination of the DNA sequence of known genes responsible for disease, identification. In addition, it is important to investigate features as methylation of the DNA molecule and examine interacting molecules such as microRNAs which are thought to regulate the timing and the number of copies made. of RNA and DNA. We are requesting funding for the Illumina HiScan system.  This is a modular system which will allow us to carry out epigenetic and genome wide association using microarray based technology as well as DNA and RNA next generation sequencing, on a single machine which will also be able to evolve with future technical developments. The HiScan system integrates a high performance scanner and an advanced fluidics device for sequencing by synthesis; this is now the most widely used method for next generation sequencing. The dual nature of the system will allow us to carry out a new range of experiments, whilst complementing currently available equipment. The Illumina HiScan will enable the analysis of Illumina microarray products and next generation sequencing experiments in a single machine: 

·  The Illumina microarrays are a glass slide based substrate upon which are bound multiple probes that can be designed to be suitable for i) genome analysis, allowing us to examine differences in the genetic structure of individuals by looking for inter-individual variation and ii) for identifying epigenetic interactions by determining the interacting molecules which modify the genetic material.

·  The HiScan reader holds a two laser system that is suitable for small RNA sequencing projects, and projects which look at sequencing known genes for novel changes. A typical RNA-seq discovery project can analyse 6 samples in 6 days whereas using current technology this might take several months.

 

 

Background

According to the online database of genetic causes of MND (http://alsod.iop.kcl.ac.uk/) there are nearly 100 known genes which may play a role in familial MND. However, these still only account for approximately 5% of the known cases of the disorder. This means a large proportion of the disease cases are of unknown aetiology. We would like to move forward our investigation of these sporadic causes by examining in greater detail a number of the underlying molecular interactions that determine the development of disease in these individuals. We have previously used gene expression microarrays to investigate the transcriptome of genetic variants in sporadic MND cases filed in our brain tissue bank. We would like to move our work forward by using more state of the art technology to carry out RNA-seq which would take expression profiling to the next level, by using next generation sequencing to provide a precise measurement of the amount of any RNA transcript expressed in the sample. RNA-seq has several advantages over microarray analysis;

i)  Detection of all RNA transcripts. RNA-seq is able to identify all RNA copies, termed transcripts, in the sample because it directly sequences all the RNA in the cell.

ii) Detection of transcript structure. RNA-seq allows new RNA structures to be identified. The publically available databases hold information from prior experiments which describe the known RNA structures. However, RNA-seq is beginning to show us that there are many variations of RNA structure to be identified and these may generate a different protein.

iii)Greater dynamic range of detection. With RNA-seq, there is no (or very low) background as sequences are examined directly and there is also no upper limit for quantification. This allows better quantification of both high and low expressed transcripts, increasing the dynamic range and five orders of magnitude of RNA species have been detected in mouse sample.

iv) Highly reproducible. Comparisons show that both technical and biological replicates are highly reproducible and the expression levels correspond to those measured either by alternative methods. We would also be able to examine the underlying DNA sequence using exome sequencing, where we sequence the regions of the DNA molecule which are the templates of particular proteins.  This would allow us to relate the actual gene and its transcribed product.

The additional advantage of the HiScan equipment is that as well as enabling us to sequence RNA and DNA, we can examine other features including 1) DNA methylation events which determine whether or not particular genes are likely to be active and 2) genome wide association studies (GWAS) to uncover novel associations of genetic polymorphisms within our patient cohort that might point us to new genetic causes of disease.

Justification for Purchasing the HiScan system

For several years genome wide association studies have provided an insight into gene polymorphisms that are linked with a disease state. The Illumina platform has been the gold standard for this type of work and contributed to a number of important discoveries (http://www.illumina.com/applications/gwas.ilmn). Recent work has indicated that epigenetic factors play an important role in the control of gene expression and may be, in part, responsible for some of the difficulties we encounter when trying to identify specific genes responsible for the disease. The Illumina methylation arrays will allow us to investigate how the genes in individuals are differentially regulated.

We have for several years examined the transcriptome of individuals using the Affymetrix GeneChip microarray system. This has enabled us to identify some important genes and pathways which play a role in neurodegeneration. However, the technology has some limitations as discussed earlier and the next generation sequencing ability of the HiScan would enable us to expand our horizons.

Rather than relying upon prepared arrays to identify known transcripts we would be able to examine the mRNA directly and not only determine the levels of expression of all genes in individuals but also whether these expressed genes are subject to alternative splicing. With the discovery that several mutant genes responsible for familial MND are involved in alternative splicing, this would enable us to pinpoint more precisely what effect these mutations are having. In addition, the sequencing capability of the machine would enable us to examine the actual exome or DNA component of specific genes to identify new mutations in genes previously discovered through the GWAS studies. This has been shown to be of importance in other areas such as schizophrenia research.

 

 

 

^{5. Finally the sequencing capability could be put to use in the field of non coding RNA identification. In another new area of study these molecules; such as miRNAs, and lnRNAs (http://www.illumina.com/products/truseq_small_rna_sample_prep_kit.ilmn), have been shown to play an important role in gene regulation and may, like methylation, be found to modulate gene expression in disease specific ways that have not yet been investigated.}

 

COST:

 

SY-103-2001 HiScanSQ System

HiScanSQ includes the HiScan Reader (SY-103-1001) and SQ Module (SY-101-2001). The HiScan system includes workstation computer, flat panel monitor, Instrument Control Software. The SQ Module includes fluidics required to perform next-generation sequencing on a HiScan. Installation and Training, and 12 months warranty (including parts and labor) are included

£267,199.00

 

SY-301-2002 cBot Cluster Generation System required for production of sequencing libraries,automated system for the generation of DNA clonal clusters by bridge amplification. Offers integrated touchscreen monitor and computer. Includes 12 months warranty (including parts and labor).

£37,538.00

SE-101-1007 Universal Starter Kit (220V) required for running of arrays Universal Starter Hardware and Software Kit. This kit is intended to manually process GoldenGate- Genotyping, Gene Expression-, MiRNA-, DASL-, Methylation-, Infinium-based products using BeadChip arrays for up to 8 beadchips of one assay type (Not intended to run more than one assay type) at a time. 220V-240V. Consumables, reagents, scanners and automation hardware sold separately.

£73,288.00

SV-103-2003 Service Contract HiScanSQ Silver for second year service contract includes full coverage on all service parts and labor. Includes comprehensive 5×24 email support and 5×18 telephone support; 3 business day average onsite response time, critical and non-critical updates, applications support, access to on-line training modules, and discounts on optional advanced training programs. Includes one Preventative Maintenance visit per year.

 

£26,975.00

 

 

SMN Replacement Gene Therapy: Novel Therapeutic Strategy for Spinal Muscular Atrophy (SMA)

 

 

SMA is the name given to the condition which is similar to MND affecting children.

 

 

Spinal muscular atrophy (SMA), a devastating inherited condition affecting children, is due to premature loss of motor neurons resulting in progressive paralysis. Motor neurons are nerve cells that send messages from the spinal cord to muscles to stimulate contraction. SMA is incurable and one of the most common genetic diseases leading to death in childhood. 50% of affected children die by the age of 2 years. Motor neuron injury is caused by deficiency of the survival motor neuron (SMN) protein. We have restored the SMN gene into cells using virus carriers (adeno-associated virus 9 or AAV9) modified to remove all their harmful properties, making them safe for human use. We successfully used our approach to restore the level of SMN protein in both skin cells, donated by an SMA child, and in a mouse model of SMA leading to markedly increased survival in the animals. These results have generated great optimism among patients and their families. Our ultimate goal is the successful translation of these proof-of-concept research data into a safe and efficacious treatment for patients with SMA. However, further studies are needed to refine our strategy before entering clinical application.

Potential benefits to patients

 

Our approach aims to replace the causative gene and this strategy can be anticipated to benefit all SMA patients by improving their life quality, by protecting motor neurons, and ultimately increasing patient survival.

Actual stages of research and development

A significant amount of work has already been undertaken to advance our approach towards clinical development. The following progress has been made so far:

 

 

a)        Remarkable pre-clinical proof-of-concept has been generated in SMNΔ7 model of SMA;

 

 

b)       Guidance has been sought from the regulatory authorities (Gene Therapy Advisory Committee (GTAC)) to ensure that our plan would deliver a product able to meet the regulatory requirements.

 

 

c)        Orphan Drug Designation has been secured from the European Medicines Agency (EMA);

 

d)       A Scientific and Clinical Advisory Board (SCAB) has been established to oversee the programme. The first meeting of SCAB was held on 8^th April 2011;

 

 

e)        The brand name for our product “ReSaGen^®” has been secured and the web-site www.resagen.com secured (website under construction).

Next steps

Given our remarkable proof-of-concept, our goal is to progress SMN replacement approach towards patient trials. Our immediate objectives are:

1) Prepare the therapeutic virus carrier at the quality acceptable for clinical use in humans;

2) Determine the minimal dose of the carrier that generates efficacy in our animal model;

3) Assess potential adverse effects in a regulatory toxicology study.

4) Secure the licensing needed to initiate human clinical trials. Funds required

So far we secured the following funds:

-       MRC DPFS (Medical Research Council development pathway funding scheme): £740,000

-       Donation of £250,000.

The funds listed above will allow us to cover the costs of objectives 1 & 2. We will therefore need to raise further funds (~£500,000) to cover the full programme. Please note that GMP vector production is very expensive and we might need multiple runs to supply enough vectors for all the pre-clinical studies listed above.

 

 

MND DNA Biobank

Sheffield is one of the three hub sites for a national initiative to generate a large well characterised resource of DNA from 1,500 MND patients with sporadic and familial disease and control cases. This resource will be invaluable for defining further genetic factors predisposing to MND and for elucidating the interaction between genes and environmental factors. The Sheffield Centre has over 800 DNA samples from MND patients and controls. This bioresource has been utilised in multiple studies investigating candidate genes as a predisposing factor for MND.

Establishing whether a high level of physical exercise is a risk factor for the development of MND
Studies in MND have been carried out to determine risk factors for the disease. As yet, no definite environmental risk factors have emerged. However, well known sportsman such as Lou Gehrig (baseball), Ezzard Charles (boxing), Donald Revey, Willie Maddren and Jimmy Johnson (football) and Jarrod Cunningham (rugby) have developed MND, leading to the strong suspicion that physical activity is a risk factor for the disease. In several studies, participation in sports/athleticism has been identified as a risk factor, although there has not been universal agreement with this conclusion.

We have recently obtained funding to establish whether a high level of physical exercise is a definite environmental risk factor for MND. This study is being undertaken in collaboration with Professor Carol Brayne, Director of Public Health University of Cambridge and Professor Nick Wareham of the MRC Biostatistics Unit, Cambridge. The initial aim is to measure the physical exercise activity of a large group of patients with MND in comparison with a group of matched control individuals, using validated instruments to measure physical exercise. In this way we can clearly identify the patients that have an unusually high exercise level.

4 Gene therapy for the childhood form of MND - spinal muscular atrophy SMA
The SITraN team, in a project led by Professor Mimoun Azzouz, have recently made a major breakthough in relation to the treatment of SMA. In this condition there is commonly a deficiency of a protein, known as SMN, and this causes motor neurone damage. Professor Azzouz has shown a dramatic benefit from using gene therapy to increase the levels of the SMN protein in a model of SMA. We are now planning to take this forward towards interventional trials in human patients.

A major programme of research, integrating clinical and laboratory studies, aimed at identifying molecular mechanisms of neurodegeneration in MND was
established in Newcastle upon Tyne and transferred to the University of Sheffield in 2000. In Sheffield, supported by programme funding from the Wellcome
Trust and the MND Association, we have established a major national Care and Research Centre for Motor Neurone Disorders.
The Sheffield MND research team is in a unique position to make a major contribution tothe search for the causes and curative treatments for MND. Research resources underpinning the programme include:

An extensive clinical database of 900 MND patients with high quality clinical details of all patients.

 The largest resource of human brain-bank material world- wide (CNS material donated by 140 MND patients), as well as DNA and cerebrospinal fluid banks.

 Robust cellular and other in vivoexperimental models of MND in which new treatment approaches can be verified.

The research team assembled has multidisciplinary skills in neurology, pathology, gene therapy, molecular genetics, protein chemistry, cell biology and pharmacology to facilitate identification of cellular targets involved in motor neurone degeneration.

MND is an excellent model for evaluating all forms of neuroprotective treatment which is why researchers at the Institute will work on finding treatments for Alzheimer, Parkinson’s and SMA which is a degenerative condition found in very young children. There will be several research programmes among which ‘Genetic susceptibility and disease modifying factors, Gene therapy, Brain reserve and stem-cell regeneration as factors determining cognitive state in Alzheimer patients'

Laboratory programmes

1 Using cellular models of motor neurone degeneration to define molecular mechanisms of neurodegeneration and to identify new therapeutic targets

We have established a robust cellular model to examine the molecular pathophysiology of motor neurone injury in the first instance in a defined subtype of MND caused by mutations in the SOD1 gene. We have used a motor neuronal cell line NSC34 cells which are a hybrid mouse motor neurone/neuroblastoma cell line, which retain the ability to proliferate whilst exhibiting many motor neuronal characteristics. NSC34 cells have been transfected with 1 of several mutant forms of human SOD1(G93A, G37R, I113T), wild type SOD1 or vector only and single cell clones derived by limiting dilution. This cell line provides a good cellular model to investigate the effects of mutant SOD1 specifically in cells with motor neurone characteristics. Using this cellular model, we have demonstrated several important insights into the cell specific toxicity produced by the mutant SOD1 protein including:

1 Biochemical changes reflecting an increased tendency to programmed cell death or apoptosis, with increased expression of cleaved caspase 9 and annexin V staining on the cell surface under basal culture conditions ;

2 Increased cell death by apoptosis, with activation of caspases 9 and 3 when the cells are oxidatively stressed;

3 Altered intracellular handling and extracellular release of superoxide and nitric oxide (NO) free radical species;

4 Increased susceptibility to toxicity from exogenous nitric oxide;

5 Altered regulation in the expression at both gene and protein levels, of components of the motor neurone internal skeleton, neurofilament light and medium;

6 The development of abnormal swollen mitochondria (which are the energy generators within cells), with impaired activity of complexes II and IV of the respiratory chain and impaired generation of energy in the form of ATP within the cells. The importance of these changes in the biochemical cascade of cellular injury produced by mutant SOD1 is reinforced by studies of apoptotic pathways, mitochondrial function and expression of cytoskeletal components in the mouse SOD1 transgenic model and in human CNS tissue.