Research activity at UCL GOS Institute of Child Health is led by Prof Nick Greene whose laboratory makes use of a range of experimental models and approaches. Within the institute and UCL we collaborate extensively with other groups with specific expertise in experimental approaches applied in our programme. These include experts in biochemistry, stem cell biology, gene therapy and other rare metabolic diseases, where we have shared interests in the underlying causes and
possible treatments. We interact closely with colleagues in the Metabolic Team at Great Ormond Street Hospital (the partner hospital of GOS Institute of Child Health).
The research programme is divided into two complementary themes. We aim:
(i) to better understand the NKH disease process, and
(ii) to use this information to develop new treatments and evaluate how well they work.
Understanding NKH – experimental models of NKH
Alongside development of treatment, we aim to better understand the disease process, which may provide new targets for treatment and give us measurable goals for testing potential new treatment.
There are two main genes whose alteration causes NKH, GLDC and AMT. As GLDC disruption is the most common cause of NKH this is the gene that we have focussed on in most of the work so far. However, the findings in these models are relevant also to AMT.
Analysis of NKH mouse models
- We have development three mouse models, with different mutations in Gldc, that have differing severity which let us study different stages of the disease.
- We have studied the changes in biochemistry that happen in brain and liver in NKH and identified metabolic alterations that were previously unrecognised in children with NKH. We have published some of this work and are working on final experiments for the next publications.
- We have investigated changes in brain development and cellular changes, both at the level of genes, proteins, cells and brain structure and also the behaviour of the mice. These findings will provide read-outs to test whether new treatments are working.
NKH cell and organoid models
In order to understand how GLDC or AMT abnormalities affect metabolism or properties of particular cells in the body we make use of models where cells are grown in the lab – this lets us do some experiments that wouldn’t be possible in a whole mouse.
1. Cell lines carrying variants of GLDC or AMT
We made changes in the NKH genes in cell lines which are related to liver and neural cells. We have been studying how this affects metabolism in the cells.
We are working with technology based on ‘induced pluripotent stem (iPS) cells that carry changes in GLDC. Some of these are made from small skin samples originally taken from children with NKH or from unaffected individuals.
These iPS cells have the special property that we can grow them in different conditions to produce specialized cells and organoids. So far we have generated liver cells and small liver organoids that are like tiny livers in a dish. We are working to describe the changes in these mini livers and at the same time we will be using them to test the gene therapy treatments.
We will also be using new techniques that allow us to grow organoids that resemble particular parts of the brain. What we find in the ‘organoid’ models we can check in the mouse models and vice versa.
Aiming for new treatment for NKH
We are following a strategy to develop gene therapy which aim to place a working copy of the GLDC gene into the body. The two projects use different technology which each have specific advantages depending on the target organs (brain and liver).
1. AAV gene therapy
We have been able to show that GLDC protein is made in the treated NKH mice in the brain and liver and to monitor glycine levels to show that the vector-produced GLDC is working. We now need to test whether different aspects of NKH are corrected; in these studies we are using several read-outs, include previously unreported changes in metabolites and gene expression that we
have identified in the mouse model of NKH. Importantly we are also analysing samples from treated mice to assess long-term safety.
2. Lentiviral gene therapy targeting the liver
This approach is particularly aimed at the liver and uses new vector technology that aims to give rapid and long-lasting reinstatement of GLDC gene function. The technology differs from the AAV approach in that the GLDC gene is inserted into the DNA, which means it won’t become ‘diluted’ as the liver grows. This work has shown a reduction in levels of glycine in the body. The next steps are planned with a series of key studies that are needed to obtain regulatory approvals.
This work will be in three main parts and in the UCL lab, we will be testing the new vectors for their effectiveness and safety in mice, and in parallel carrying out treatment of liver organoids to test how the vector acts in human cells.
3. Treatment to control glycine levels
We have been working to test whether glycine levels could be lowered using treatment with cinnamate, as an alternative to benzoate which has unpleasant side-effects. We treated mice with cinnamate and have carried out for detailed analysis of the biochemical changes brought about by cinnamate and benzoate and we are working through analysis of the data. Initial analysis confirms our previous findings showing a beneficial effect of cinnamate.