Generation of a site directed gene integration platform for induced pluripotent stem cell lines.

Lead Research Organisation: Aston University
Department Name: Sch of Life and Health Sciences


To understand how the human brain works and therefore how to intervene when it goes wrong we need to be able to study brain cells such as neurons. Living human neurons are virtually impossible to obtain in order to understand how basic cellular processes occur in the human brain. Stem cells offer the ability to produce human neurons and other brain cells to use in such studies. Recently there has been a revolution in the field of stem cell research. Now it is possible to take skin cells from disease patients, transform them into stem cells, and then from these generate patient specific neuronal networks in culture.

The ability to insert novel genes into neurons is of great importance and allows scientists to study cell behaviour in response to different triggers such as gene mutations, but also allows them to control the activity of cells. However this is particularly difficult in cells such as neurons, as they do not divide, and often requires specialised reagents such as viruses, which are used as vectors to insert genes into non-dividing cells. When using viruses or other common insertion methods, these genes are often inserted randomly into the cells own DNA and if inserted in the wrong area, can alter the ability of the cell to carry out its normal functions. One solution is to modify the genome of the stem cell to incorporate a region of DNA that can serve as a 'docking site' or 'launch pad' for novel genes that is in a safe area. In this way novel genes will always be incorporated into the same place in the cells genome without altering the cells normal functions. As a stem cell can be made to differentiate into many different types of cell, creation of a stem cell with a docking site for gene insertion will provide a platform for researchers to assess the function of inserted genes in many cell types.

In this project we propose to combine the skills of Axol biosciences, a company who specialises in providing stem cell derived neurons to all researchers with the skills of the Neural stem cell group at AU to create such a platform stem cell line. The platform line will be tested by the insertion of genes encoding Channelrhodopsin (ChR) proteins, which will make the neurons sensitive to laser light, allowing researchers to non-invasively stimulate neurons into the types of activity that are observed in the brain. This is a technically difficult challenge and will develop the skills of both partners. Scientists from Axol will also be trained within Dr Rhein Parri's lab in techniques that will enable them to test their cell lines to ensure that they behave as cells do within the brain. This work will provide highly specialised scientists with the unique combination of skills in both fields that will enhance their research capabilities. The experience of both interchangers in both academic and commercial environments will build new skills within each organisation that will be embedded in future research and development to produce future leaders in this area of research.

Planned Impact

The number of people over the age of 65 in the UK has now risen to over 10 million, with a concomitant rise in age related disease and care costs, dementia care costs more than that of cancer and heart disease combined. Basic research in neuroscience has the capacity to improve public health, by providing new targets and models to test and improve pharmaceutical interventions for the treatment of neurodegenerative disorders. Development of technologies to facilitate such research, along with the training and knowledge gained in this FLIP will have far reaching benefits to a wide group of beneficiaries.

Who will benefit?

AU, Axol Biosciences, Neuroscience/research community, Pharma and Biotech companies, Economy, Students, NHS Budgets, Government, patients and their carers, The Public.

How will they benefit?

Aston and its collaborators will immediately benefit from the training and skills acquired, along with access to the platform line. Other researchers will then have access to the quality controlled platform line (2 yrs) providing a tailored gene expression system to easily study proteins/genes of interest in cutting edge systems. This will reduce start up times for new projects and remove the technical challenges which preclude many researchers from the adoption of such technologies.

Axol Bioscience
Within year one of the grant Axol will benefit though acquisition of electrophysiology skills to enhance their QC procedures, and research. They will also benefit through development of a unique product line (2 yrs) which has been developed in conjunction with users and is amenable to the needs of most researchers. The line will also be compatible with other cell lineages as Axol grow and diversify their product base (3-10 yrs). They will also benefit through an enhanced understanding of their customer base.

Pharma and biotech companies
The adoption, of a commercially available neuronal model capable of selective gene expression will have a knock-on effect to other SME's. Functional neuronal models and reporter cell lines will require cell based reagents as well as neurophysiology platforms such as MEAs, imaging platforms and microscopes. Hence increasing demand for such products benefiting SMEs supplying them.

Growing numbers of elderly people increase the pressure on NHS budgets. Dementia costs the UK economy £23 billion a year. This FLIP will provide an easily manipulatable iPSC platform line and complex neuronal models amenable to controlled gene expression. This will provide a powerful tool to facilitate the scientific discoveries to inform the development of new treatments aimed at ameliorating neurological disease (5-15 yrs).

Within the lifetime of the grant (1-2 yrs), practical examples of such cutting edge technologies, can be incorporated into undergraduate and MSc lectures at AU. Producing graduates prepared for their future careers by ensuring they are informed of the latest scientific developments, and inspiring students at a crucial stage to consider a career in brain research.

This project will highlight the benefit of investing in high quality research and training that can provide longer term economic benefits through both industrial progress (3-5 yrs) and healthcare benefits (5-15 yrs).

There is growing public interest in the functioning of the brain and the use of stem cells. Disseminating the findings of this research (1-2 yrs) in line with our existing commitment to public engagement will increase public understanding and appreciation of such cutting edge research.

In the medium to long term (3-5 yrs) this project has great potential to positively impact the UK economy, increasing the global economic competitiveness of a vibrant UK based start-up company, and ultimately providing a resource for UK based scientific discovery.


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Description We developed the constructs and expertise to routinely target the genome of stem cells in the same location to insert DNA capable of expressing proteins not normally expressed in these cells or their differentiated counterparts. This enables the creation of differentiated cells such as neurons which express molecules that can report upon neuronal activity. In addition, training provided by the award has enabled the routine creation and maintenance of induced pluripotent stem cells at Aston along with the ability to genome edit these cells in other locations enabling us to create disease models and other tools for future research. The training and collaborative nature of this grant between Aston University and Axol Biosciences has developed our research relationship and directly enabled the successful application for the 3.2 million Euro EU funded FET MesoBrain consortium. Indeed the skills and knowledge acquired by Dr Nagel as key beneficiary of the FLIP award enabled him to contribute fundamentally to the Meso Brain application on which he was a Co-Applicant.
Exploitation Route Currently we are utilising the platform constructs to build reporter lines for use by us and members of the European funded FET Meso-Brain consortium. We are continuing to develop the platform line and when the initial problems are resolved with satisfactory performance, the line will be made available via Axol Biosciences as originally described as will reporter constructs for use with the line. After publication of work involving reporter lines in the FET Meso-Brain consortium, base targetting constructs and reporter constructs will be made available to the research community for creation of their own derivatives of such lines.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Future Emerging Technologies
Amount € 3,225,891 (EUR)
Funding ID 713140 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 09/2016 
End 08/2019
Title AAVS1 targetting constructs 
Description we have developed quick change targetting constructs for routine genome editing of the AAVS1 site for the development of neuronal reporter lines. Whilst the RMCA line is not yet fully functional, these constructs were designed in such a way to allow different genes/promoters to be inserted in a single reaction enabling more rapid genome editing of different cell lines. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Knowledge gained enabled vision and writing of successful Meso-Brain application. 
Description MesoBrain 
Organisation University of Barcelona
Country Spain 
Sector Academic/University 
PI Contribution Part of EU funded Meso Brain consortium FET award. Helped write grant. Participated in research programme.
Collaborator Contribution Different parttners contributed different expertise from photonics, physics, imaging.
Impact No outputs yet.
Start Year 2016
Description Guest Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Dr Nagel has used his gained experience to provide guest lectures to both undergraduate Biomedical Science and Postgraduate Stem Cell masters students at Aston University. Outcomes include an increase in requests by these students to conduct research projects in our laboratory.
Year(s) Of Engagement Activity 2015,2016