NKH Research Updates
Nonketotic Hyperglycinemia (NKH) Research Updates
We’re pleased to be supporting the gene therapy work at UCL GOSH Institute of Child Health led by Prof Nick Greene. They are experts in their field, and we firmly believe they are the closest to bringing about an effective treatment for children with NKH and their families.
The team at UCL are following a strategy to develop gene therapy which aim to place a working copy of an NKH gene into the body, they have two projects on the in progress (the brain and liver gene therapy projects) which the Mikaere Foundation grants support.
You can read more on the UCL NKH website at ucl.org.uk
We also support the work done by Prof. Johan van Hove at the University of Colorado. Prof van Hove has been researching NKH for over 30 years, and his work is cited in almost every research paper related to NKH. Prof. van Hove’s team are working on an effective treatment that may be used in conjunction with the treatments being explored at UCL. For more information visit: cuanschutz.edu
If you are a researcher involved in projects with NKH, and would like to put yourself forward for funding, please fill out a Request for Funds Form and a Due Diligence Form, and return to hello@mikaerefoundation.org to be considered.
Update date: June 2024
UCL Updated Research Paper on NKH Gene therapy and AMT gene therapy research started
Highlights:
– Creating more complex GLDC mouse models
– AMT work has started, both in growing cell models, and in gene therapy research
– UCL has identified metabolic alterations that were previously unrecognised in children with NKH (research paper on it’s way)
– Cinnamate studies have been positive, and a research paper is underway to share these results.
– Discovered the structural brain malformations are due to folate metabolism, and not high levels of glycine. The UCL team previously have found protective effects of formate and methionine (published in 2015 and 2017). They are using this system to test other molecules that we would then aim to test in the NKH mice.
Overall strategy
The UCL research strategy continues to focus on two main themes: They 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 provide measurable goals for testing new treatments.
Among the genes whose alteration causes NKH, the focus has largely been on GLDC so far but we
have now also begun to work on AMT.
Analysis of NKH mouse models
UCL are using several different mouse models, with different mutations in GLDC. These have
differing severity which allow the NKH Research team to study different stages of the disease. A further model is currently in production with the aim to identify and track the cells that should make GLDC but don’t. This
is quite a complex model to make but it should be very useful.
UCL 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. Over the last few months, work has been carried out to examine gene expression changes that correlate with metabolite changes. There is a paper in progress to report this data.
A major piece of work over the last year has been a project to carry out very detailed analysis (at a single cell resolution) of gene expression changes in the brain. In the first instance the team have focused on a specific part of the brain and a new post-doc joined the group in December 2023 who is leading this work. Her aim is to ask if the cellular composition is altered and/or whether particular cell types have specific changes in gene expression that may reveal where targeted treatment is needed.
The UCLE team 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 – this is being followed up with behavioural changes to test if they give reliable readouts for testing therapy.
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.
- Cell lines carrying variants of GLDC or AMT
Some changes were made in the NKH genes in cell lines which are related to liver and neural cells. The team has been studying how this affects metabolism in the cells and have identified some previously
unrecognised biochemical changes which is now being examined in various model systems - Organoids generated from induced pluripotent stem (iPS) cells.
In a previous update it was shared how work to make liver cells and liver organoids from cells that have changes in GLDC. This has been expanded the work to a parallel project in which a PhD student is investigating cells with changes in AMT. In both cases the team are asking about the effect on liver organoid
development and on the early stages of nervous system development. The liver organoids have also recently been used in safety testing in the gene therapy projects.
Towards new treatments for NKH
The UCL Team are aiming to develop gene therapy to place a working copy of the GLDC gene into the body. Two projects use different technology which each have specific advantages depending on the target organs (brain and liver). A third project, using a different gene therapy approach has received funding and we aim to being this project shortly. Finally, we have initiated work on a fourth project aiming to develop gene therapy for NKH caused by alterations in the AMT gene.
- AAV gene therapy for GLDC
The UCL team 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. They showed
that glycine levels are lowered in the blood and brain and that folate metabolism is normalized.
They found some previously unreported changes in metabolites (eg. choline and betaine) and
correction of these in treated mice. The team also generated vectors that make the human GLDC
protein. This work was recently published https://doi.org/10.1016/j.ymgme.2024.108496 and is
open access so anyone can read it.
The UCL team have already started a new set of experiments to treat further cohorts of mice to test
treatment combinations and test whether different aspects of NKH are corrected using a wider
range of read-outs that we have now identified. The next steps for this project are to transition to
manufacture and testing of clinical-grade vector and safety testing. - 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. The UCL Team completed a large project to test whether the treatment lowers glycine in
the blood and body tissue, to correlate this with the amount of gene therapy vector that was
successfully inserted and with the amount of GLDC protein (from the vector). They also carried out
treatment of liver organoids to test how the vector acts in human cells.
The next steps of the project to obtain regulatory approvals include production of clinical grade
vectors (out-sourced), testing for their effectiveness and safety in mice (in my lab and out-
sourced), and planning for clinical trial. The project is currently awaiting go ahead from the funder. - Treatment to control glycine levels
The UCL 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. They treated mice with
cinnamate and have carried out for detailed analysis of the biochemical changes brought about by
cinnamate and benzoate. The team confirmed a beneficial effect of cinnamate on glycine. Other effects
of benzoate and cinnamate show differences between these two treatments and we are working
to finish a paper describing all these effects. As part of this work we are analysing, the results to
see if there are effects of benzoate which are unexpected and which could be avoided. - Small molecules for normalization of folate one-carbon metabolism
In parallel with studies on the NKH mouse model, other projects are examining the effects of GLDC
or AMT loss of function during embryonic development, because we know that this can cause
structural anomalies such as neural tube defects.
The UCLe team previously found that the structural changes in the embryonic brain result from suppression of folate metabolism and not excess glycine. Therefore, in addition to understanding how these
conditions arise, the embryo system enables us to test possible treatments for correction of folate
metabolism with a very clear read-out of preventing neural tube defects. For example, they have
found protective effects of formate and methionine (published in 2015 and 2017). They are using
this system to test other molecules that we would then aim to test in the NKH mice. - AMT
The UCL team have initiated projects to develop models for NKH caused by abnormal AMT gene. They already have cell lines with absent AMT, and have made 3 mouse models which are now being
characterised. The AAV vectors to test in these mice have already been optimised so its everyones hope this
project will move forward quickly.
Update date: May 2024
Discovery of two compounds that may impact glycine levels in the brain.
Overview
- Dr Johan van Hove and Michael Swanson (working as a team at the University of Colorado, in Denver).
- They have multiple mouse models with NKH (GLDC)
- Dr Johan van Hove has discovered two compounds which, it’s hoped, have the potential to:
- Reduce glycine levels directly in the brain
- Help with folate metabolism in the brain (something that is impaired currently in NKH. The Glycine Cleavage System (GCS) process helps supply the brain with one carbon folate molecules, which are missing when the GCS is broken).
- The potential impact of this could change the severity and course of NKH and have a drastic impact on the quality of life for children diagnosed with NKH moving forward.
Update date: Dec 2023
UCL Published Research on NKH Gene therapy
In December the first ever paper about NKH gene therapy was published, showing via mice models that gene therapy is a viable cure for NKH. Clinical trials look to be 3-5 years away (or longer). You can read the paper here: https://www.biorxiv.org/content/10.1101/2023.12.15.571844v1
Takeaways include:
– The research team used gene therapy to introduce a working GLDC gene into both brain and liver cells of mice with NKH (so, before gene therapy the mice didn’t have genes that could build proteins that are part of the glycine cleavage system, meaning they couldn’t process glycine)
– They added both mice genes and human genes, and both worked well to express the gene protein
– They used an AAV vector (which is a virus that targets a cell – similar to whats used in the covid vaccine) to get the gene into the cell.
– The cells in both the brain and the liver were able to show GLDC protein expression meaning the added GLDC gene was working as it should.
– With liver treated cells, mice had lower blood glycine levels.
– In mice where they only treated brain cells, blood glycine levels did not normalise (as expected – because it’s the liver that manages blood glycine levels, not the brain). They did see lower glycine levels in brain tissue though, which means glycine levels in the brain can still be lowered even if blood glycine levels are high (!)
– We know the one carbon folate supply is severely diminished when the glycine cleavage system is broken, this is normalised in the brain with the treated mice.
Update date: Feb 2023
Non-Ketotic Hyperglycinemia – Research Strategy at UCL (GOS Institute of Child Health)
OVERALL STRATEGY
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.
2. Organoids
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.