A Multi-omics Investigation of Ring14

Awardee: Heather Mefford

Institution: St. Jude Children's Research Hospital

Grant Amount: $67,897.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

Ring14 is an understudied and life-limiting rare chromosomal disorder with symptoms including severe and drug-resistant epilepsy, slow growth, low muscle tone, intellectual disability, and other multi-system health issues. For the last 10 years, the major hurdle in studying Ring14 has been the lack of a reliable model that accurately represents what is seen in individuals with Ring14 due to the unique structure and instability of the ring chromosome. Additionally, cellular models for neurological conditions do not always mirror the true biological state of the brain. Spatial transcriptomics is a new technology that combines brain imaging, gene sequencing, and cell characterization simultaneously to provide holistic biologic insight. In this collaborative study, we will perform spatial transcriptomics in brain tissue to directly and simultaneously study how Ring14 affects the cellular structure, organization, and gene expression in the brain. We will also study methylation – a biomarker for the regulation or turning on/off of genes – in multiple Ring14 patients and compare their methylation patterns with individuals with other chromosome 14 differences (i.e., deletions) and healthy individuals without epilepsy. Previous studies and karyotypes (chromosome genetic tests) have shown that the instability of the ring chromosome causes mosaicism in the body, meaning the ring chromosome is not present in all cells. So far, no study has been able to assess the extent of mosaicism in the most disease-relevant tissue, the brain. By evaluating the content of ring14 in brain tissue and integrating methylation and transcriptomic data, we will elucidate the unique biology and mechanisms of Ring14. Our study has the potential to inform future targeted therapeutic development for this devastating life-limiting condition.

Novel Adeno-associated viral vector (AAV) mediated RNA editing treatment for KCNT1 epilepsy

Awardee: Rajvinder Karda

Institution: University College London

Grant Amount: $70,619.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

KCNT1 epilepsy is a severe childhood genetic epilepsy, which leads to life-long disability. Spelling mistakes, or mutations, in the genetic code of KCNT1 cause epilepsy of infancy with migrating focal seizures (EIMFS). EIMFS becomes apparent in the first 6 months of life, where babies present with frequent seizures, developmental delay, and movement disorders. Sadly, patients also have an increased chance of premature death. The KCNT1 gene codes for a potassium channel which changes nerve cell (neuron) excitability. Mutations associated with this form of epilepsy result in increased channel activity in brain cells, making them more excitable. Current drug treatments are unfortunately inadequate and ineffective. The process of making proteins in cells involves translating DNA (the genetic code) into RNA (the protein code) which is then made into proteins such as the KCNT1 channel. We aim to develop a novel RNA editing therapy treatment for EIMFS, altering the protein code so less protein is made. We will deliver the RNA treatment within a virus called adeno-associated virus (AAV). When the AAV enters neurons, it will reduce the amount of KCNT1 protein and normalise the channel activity. We will test this new treatment in a Kcnt1 mouse model which has an over-active KCNT1 channel and seizures very similar to those seen in patients. We will also test the treatment in neurons made from skin cells donated by patients with KCNT1 epilepsy. In the future we hope this could be developed into a treatment for patients. Although like other gene therapies it would need to be delivered to the brain, our treatment would have several advantages including being a one-off treatment unlike other RNA treatments which require repeated spinal taps or lumbar punctures. Therefore, in this proof-of-concept study we will develop and test a novel RNA editing one-off treatment to improve KCNT1-epilepsy.

SKIP-AT: a comprehensive, systematic platform approach for targeted exon skipping of ATM

Awardee: Matthis Synofzik

Institution: Hertie-Institute for Clinical Brain Research, University of Tübingen, Germany

Grant Amount: $53,240.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

We will develop and validate a systematic, comprehensive platform approach for targeted exon skipping of constitutive ATM exons. For this, we can directly leverage and extend our established, scalable platform for the development of patient-specific antisense oligonucleotides (ASOs) for ATM - with already proven efficacy from preclinical to clinical translation in our hands. Our ATM exon-skipping approach will demonstrate and validate a systematic experimental path that will allow to provide the genomic medicine field with the urgently warranted comprehensive knowledge on which exons of the ATM gene can be (ASO-)skipped - and in fact even allows to already prepare the most promising skippable exons for clinical ASO treatment development.

CSF dynamics in mucopolysaccharidosis type I

Awardee: Igor Nestrasil

Institution: University of Minnesota

Grant Amount: $60,655.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

This project aims to measure the motion of the cerebrospinal fluid (CSF) through the brain with magnetic resonance imaging (MRI) and associate these measures with disease severity in mucopolysaccharidosis type I (MPS I). The findings may determine whether CSF motion is impaired in MPS I with implications for the disease severity or the drug distribution administered to the intrathecal space (space surrounding the spinal cord) or subarachnoid space (space around the brain).

To Study the potential for Collagen Mimetic Peptides to reduce progressive damage to the Bruch's-RPE-Photoreceptor Complex in Choroideremia.

Awardee: Malia Edwards

Institution: Johns Hopkins University

Grant Amount: $64,735.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

Collagen is the most common protein in the human. It is found in all organs in either its structural form or as a major component of the extracellular matrix. The source of newly formed collagen arising during tissue growth and remodeling is the nearby cells, often mesenchymal but also epithelial. A healthy body undergoes collagen remodeling on a constant basis, but aging or gene deficiencies can cause the ability to produce replacement collagen to decrease. In certain disease states collagen-producing cells are often involved and may become damaged as well, leading to a reduction in collagen replacement which slows the tissue healing process. A therapeutic with the ability to repair damaged collagen can facilitate recovery and halt progressive damage to critical tissues and organs, like the eye, when it is subjected to such diseases. CMPs are vastly different from denatured collagen supplements, which are highly advertised nutraceutical products and which do not have the direct collagen repairative activities associated with CMPs. CMPs have been shown to be efficacious and safe in humans when applied topically as eyedrops and systemically in animals under FDA regulated toxicity studies.

Leveraging predictive models to design high-throughput assays to resolve variants of uncertain significance (VUS) in SYNGAP1

Awardee: Gemma Carvill

Institution: Northwestern University

Grant Amount: $61,222.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

SYNGAP1-related disorder is caused by pathogenic (disease-causing) loss of function variants, and while most variants described to date are truncations, there are also at least 50 pathogenic missense variants that have been described. However, missense variants are more likely to be classified as uncertain significance i.e. of unknown impact on protein function, we call these VUS. This is because of the difficulties with predicting whether these variants impact SYNGAP1 function using current tools. Here we will use a computational approach to determine what characteristics of missense variants are more likely to impact SYNGAP1 function. We will then design and test a method/assay based on this characteristic to identify pathogenic missense variants. This method will be able to screen hundreds of missense variants at a time. We will make this data available in an accessible manner to the patient community.

Identification of vulnerable cell types and quantification of cell type-specific differential gene expression in the Pitt-Hopkins Syndrome mouse model

Awardee: Brady Maher

Institution: Johns Hopkins University School of Medicine

Grant Amount: $73,473.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

The brain is composed of two major cell types, neurons and glia; however, these two cell classes quickly become very complex as you begin to categorize them into specific subtypes of cells based on their structure and function. For instance, glia cells are comprised of astrocytes, oligodendrocytes, and microglia, which all have unique structures and functions, and these three cell types can be further divided into additional subtypes. This complexity is even greater for neurons, which are first subdivided into excitatory and inhibitory neurons, but then have so many subtypes they cannot all be listed here. Recent technological advances in single cell sequencing (scRNAseq) have emerged that allow us to quantify the expression of genes within individual cell types, which is critically important because each subtype of cell expresses different genes that regulate the specific function of that cell type. Transcription factor 4 (Tcf4), is a transcription factor that regulates expression of specific genes, however the set of genes it regulates, differs depending on the cell type. Currently, there is no data available that describes how disease-causing mutations in TCF4 effects cell type specific expression in the brain. Therefore, we propose to perform scRNAseq on brain samples from the PTHS mouse model and WT littermates. Previous results from our lab and others have shown that Tcf4 is highly expressed during brain development and mutations in Tcf4 result in alterations in the proportions of specific types of cells (e.g., GABAergic interneurons and oligodendrocytes), however we hypothesize this list of vulnerable cell types is not complete. In addition, Tcf4 is expressed throughout the lifespan, where it is continually regulating gene expression in a cell-type dependent manner. Unfortunately, the field currently lacks an understanding of which sets of genes Tcf4 regulates in any specific cell type. Our proposal is designed to fill in these important knowledge gaps and will help to confirm prior observed vulnerable cell types and identify new cell types of risk. In addition, our approach will identify genes that are differentially expressed in specific cell types, which will help to identify genes that can be targeted by cell type-specific therapeutic approaches. We plan to openly share our results with the research community. We believe it will be beneficial to improving emerging gene therapy approaches by informing us about which cell types should be modified. We believe it will serve to identify vulnerable cell types that may in the future be replaced using cell replacement therapies. Lastly, we believe these datasets will be useful for computational approaches that are using artificial intelligence and machine learning paradigms to identify therapeutic targets and predict effective therapeutic compounds.

Unravelling HGprt functioning in fore- and midbrain development using brain organoids

Awardee: Sharon Kolk

Institution: Radboud University, Donders Institute for Neuroscience

Grant Amount: $68,367.00

Funding Period: February 1, 2024 - January 31, 2025

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Summary:

LND is an incapacitating disease characterized by a neurobehavioral phenotype, cognitive deficits and self-injurious behavior caused by HPRT1 gene mutation(s). LND is specifically associated with a reduction of dopamine in the brain. Recently, we reported that proliferation and migration patterns of developing midbrain dopamine (mDA) neurons are disrupted in absence of the gene, resulting in abnormal brain development in an LND animal model. To dissect the role of the causative gene in human fore- and midbrain development, we intend to investigate human-specific brain organoids using patient-derived induced pluripotent stem cell (iPSC) lines carrying HGPRT loss-of-function (LOF) mutations, age-matched control lines as well as edited control lines carrying HGPRT mutations. In addition, assembloids -fusions of dorsal forebrain organoids and midbrain organoids- will be generated in various combinations of control versus LOF tissue at multiple time points. It is furthermore shown that folic acid plays a role in purine metabolism and that physiological levels lead to metabolite accumulation in LND patients. Therefore, we will add various concentrations to the culture medium of both the fore-as well as the midbrain organoids at multiple developmental timepoints. Eventually this will give us insight into the developmental time window where we can, either genetically or pharmacologically, intervene in the future to alleviate particularly the behavioral and/or cognitive characteristics associated with LND.