University College London
Mouse Modelling + NKH Progression
By using mice that have an NKH genetic alteration we can collect samples to look in detail at the early stages of NKH progression. By understanding which cells are affected and the metabolic and molecular changes that cause these effects, theories for treatment can be developed.
Metabolic/Mass Spectrometry Analysis
Metabolic/Mass Spectrometry Analysis provides an insight into what chemicals are altered in the brain with NKH. These neurological studies allow us to ask test the effects of these metabolic changes. By using EEGs to test whether the mice show features of epilepsy and examining brain slices in great detail with staining for different types of cells and cell damage, we’ll understand what areas of the brain are affected by NKH, and the early progression.
Small molecule/drug approaches
Looking at possible treatment options, and whether they have an effect on the progression of NKH.
Conditional Genetic Rescue
An NKH mouse has a piece of DNA called a ‘gene-trap’ that turns off the GLDC gene. We can use a genetic trick to inactivate the gene-trap so that the gene is turned back on. We want to ask (1) whether turning the gene back on after birth is sufficient to prevent the disease and (2) if is sufficient to just turn the gene back on in particular parts of the body.
- Test a gene therapy vector in the mice and we are working with a group at UCL who have done this in other metabolic disease.
- Using other models such as cell lines (where we could look at human cells).
- Expansion of work with the zebrafish model that we’re trying to generate (which would be good for drug screening).
Boler-Parseghian Centre for Rare and Neglected Diseases
University of Notre Dame, Prof. Suhail Alam
The ND–NKH Program
Through a Notre Dame Student education program, NKH medical records are collected and used to develop a natural history of NKH – essentially how NKH unfolds and progresses. This data will hopefully provide an NKH roadmap for the different mutations, so we can understand what future expectations and symptoms a child with NKH might have. It also provides researchers a way to identify whether treatments are working as expected.
The Molecular Study
Through skin biopsies and blood tests researchers are able to evaluate the molecular and cellular factors that contribute to NKH. This in turn will be used to develop targeted therapies and treatments for NKH.
Drug Therapy for NKH Enzyme Deficiency
NKH causes a faulty enzyme function – the body is unable to process glycine. In most cases this is because the faulty enzyme is mutated – it’s unable to take on the normal shape, which means it’s not recognised by the processing enzyme.
For example, the normal enzyme in figure one (a p protein) converts it’s substrate (blue circle) correctly. When there is a mutation, the process can no longer function correctly because of the mutated shape.
The goal is to find drugs that can help the P protein take on its shape and restore normal function. Hundreds of thousands of molecules are screened to find a few that can alter the P protein into a functional shape..
To do this engineered antibodies are developed – mutated cells are grown in a clinical lab. Drug molecules from chemical/drug libraries of Eli Lilly & Co (a pharmaceutical company) are added to the mutated cells. When the drug molecule helps the mutant protein return to the functional 3d form, the antibody will bind to the enzyme. This binding will trigger a signal that is captured by a microscope camera, identifying a new potential drug hit. These hits will be converted into leads, tested for efficacy and safety and hopefully developed as new drugs for NKH.
University of Colorado
Department of Pediatrics, Dr Johan Van Hove
NKH Genes and Outcome Severity Study
It’s thought that DNA sequence mutations are a primary indicator of NKH outcome and severity. To confirm this, the impact of different genetic NKH mutations on protein stability and function are examined.
This involves creating a mutation in a genetic construct, expressing and assaying the enzyme to see if it has any activity left. The enzyme has multiple components (each of which we have to make) and active groups like lipoic acid (which we have to attach). Although, this is a technically challenging project, I am happy to report that we are making substantial progress. A fabulous postdoctoral worker in the lab, Heather Szerlong, has made all the components and is now working on optimizing the assay.
In terms of clinical relevance, it seems that certain therapeutic strategies are more likely to work in patients who have specific mutations. Improved understanding of the pathological effects of mutations in NKH is an essential prerequisite for a “personalized medicine” approach, which can significantly improve the outcome for select patients with this disease.
We have identified a subset of patients (about 5%) with NKH who have defects in new genes not previously associated with this NKH. We have identified several of these genes, and found that they all impair the synthesis of lipoic acid.
We are currently working on generating the final conclusive evidence that we have found the correct genes. An exciting aspect of this work is that it has led us to the generation of a novel treatment strategy for this new form of NKH.
We want to work on the disease process that makes children ill when they have NKH. Only when we understand how this process works, can we have a rational basis for novel therapies. This project works in two ways: first, we will analyze information from human patients with NKH, and second, we will genetically engineer a number of mouse models with NKH. The first mouse with NKH is currently under construction, and if everything goes smoothly we anticipate having mice with NKH to study in 6 to 12 months. Having a mouse model is very important because we can look at what makes the brain sick, and we can test treatment strategies on the mice in a way that is impossible to do in humans.