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: October 2024

Gene therapy exploring vector production and safety studies, and RNA treatment study launched!

Highlights:
  • Significant progress has been made on understanding NKH in GLDC mice models, particularly with a detailed analysis of gene expression changes in the brain and in brain structure.
  • There are now 5 AMT mouse models, and UCL are checking biomarkers, so they can be used in treatment studies.
  • GLDC gene therapy in both the brain and liver continues, and are in the process of testing vector production and obtaining regulatory approvals.
  • RNA treatment study launched
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

Analysis of experimental models of NKH continues to be a major activity, aiming to both understand the disease process (including identification of new targets for treatment) and to provide outcome measures to evaluate treatments, across both AMT and GLDC.

Analysis of NKH mouse models

GLDC

  • UCL have continued with several projects analysing our two main mouse models which have impaired function of GLDC (the main gene affected in NKH).
  • They have adapted their models to allow rescue or knockout of GLDC function at selected times and in selected tissues. They previously used this to rescue GLDC expression just in the liver (published in 2020) and have more recently again used this approach to rescue expression in selected cell types in the brain. These experiments allow ‘proof of principle’ for gene therapy approaches by telling us which tissues they should prioritise for gene replacement.
  • UCL have made significant progress with their project which has involved detailed analysis of gene expression changes in the brain, in combination with detailed analysis of brain structure. Current work is focused on validating the gene expression changes that they have found and evaluating the significance of these findings in the disease.
  • In their previous report they highlighted a study which has examined changes in biochemistry that happen in brain and liver in NKH, and which identified metabolic alterations that were previously unrecognised in children with NKH. The paper to report this data is in progress.

AMT

  • AMT is the causative gene in about 1/5 of children with NKH. They have now generated 3 mouse models with differing changes in the AMT gene and imported a further 2 models that were generated elsewhere. They are establishing these mice at UCL and have begun to characterise them as models of AMT-related NKH. The mouse strains will be an important resource for testing new treatments and for understanding how and why changes in AMT lead to NKH.
  • They now have access to mouse models which carry AMT gene changes that are found in children with NKH. UCL are now checking the effect on biomarkers such as glycine. They also have two models in which the AMT is completely disrupted. In one of these they have replaced the protein-coding part of the gene with the sequence for a fluorescent protein – enabling them to track AMT-knockout cells.
NKH cell and organoid models
  • Work with the tissues and organoids derived from induced pluripotent stem (iPS) cells has continued in projects being carried out by two PhD students. The aims are to analyse effects of AMT and GLDC loss of function in various human cell types with the current focus on nervous system and liver cells.
Towards new treatments for NKH

UCL continue to work on several different projects with two main strategies which comprise, (i) gene therapy (one-time treatment) and (ii) other treatments which aim to lower glycine and/or improve folate metabolism.

Gene therapy

These projects aim to place a working copy of the GLDC or AMT gene into the body. The two main GLDC projects aim to target either the brain and liver (using an AAV vector), or primarily the liver (using a lentiviral vector). UCL showed that both these approaches effectively lower glycine.

The activity over recent months has involved planning the next steps in moving the work from mouse models towards clinical trial. This will need scale-up and production of a ‘clinical-grade’ vector (in a specialist facility), confirmation that this vector is active, and safety testing as required by regulatory bodies before a clinical trial can be carried out.

1. AAV gene therapy for GLDC

UCL have identified a production facility and developed a plan for pilot work to test feasibility of vector production – they have completed initial steps in our lab and the out-sourced work is starting this month. In parallel, they are preparing a major grant application for funding to support the next phase of the project (manufacture, safety etc). UCL have now completed a set of experiments to test whether AAV gene therapy could be used together with other glycine lowering approaches (e.g. benzoate).

2. Lentiviral gene therapy for GLDC (targeting the liver)

UCL have planned the next steps of the project to obtain regulatory approvals including production of clinical grade vectors, testing their effectiveness and safety, and planning for a clinical trial. Since the last report they have completed the deep molecular biology analysis of treated human liver organoids which has provided important information to support safety.  

3. AAV gene therapy for AMT

UCL have made good progress on the new project aiming to develop gene therapy for NKH caused by mutation of the AMT gene. They tested vectors in cells and production of vector for treatment of the AMT mouse model(s) is in progress.

Additional approaches for development of new therapies

UCL are testing several different approaches using either small molecules, nutrients or drugs which may prevent or correct specific features of NKH. This includes studies on using cinnamate and folate-related nutrients.

Cinnamate treatment to control glycine levels

UCL are working to finish a paper describing effects of benzoate and cinnamate – both of which can lower glycine in the NKH (GLDC) mouse model. Additional current work is in progress to repeat the key biochemical tests using alternative methodology to validate the findings

Small molecules for normalization of folate one-carbon metabolism

Testing of folate-related molecules is ongoing in the embryo model, building on their previous findings of protective effects of formate or methionine. This work initiated was in the GLDC mouse models and we hope to extend this to the AMT models soon.

RNA-based treatment for NKH?

UCL would particularly like to highlight a new project for which we obtained funding from the MRC with additional support from the Mikaere Foundation. This project aims to develop an RNA-based treatment, which may be beneficial – as it can be used alongside Gene Therapy and has the potential for repeated dosing.

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.

  1. 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
  2. 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.

  1. 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.
  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. 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.
  3. 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.
  4. 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.
  5. 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.

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