Targeted reactivation of ZC4H2 using miniature Cas editors

Awardee: Julian Halmai

Institution: University of California, Davis

Grant Amount: $46,846.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

The overall goal of this proposal is to develop CRISPR therapeutics for ZC4H2 Associated Rare Disorders. If successful, reactivation of the X-linked ZC4H2 could result in rescue of disease associated phenotypes in female patients affected by the disorder. This proposal utilizes AAV ready miniature epigenome editors that have great potential to improve the field of extant CRISPR therapeutics.

Structural and Functional Investigation of the ZC4H2 Protein

Awardee: Fahad Ali

Institution: Mohammed Bin Rashid University Of Medicine and Health Sciences

Grant Amount: $46,846.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

ZC4H2 Associated Rare Disorders (ZARD) is a rare genetic disorder that impacts the nervous system. It is caused by mutations within the ZC4H2 gene. ZC4H2 codes for a protein that, as of yet, has not been studied and is of unknown function. In this project, we aim to investigate this function using a combination of cellular and molecular approaches to guide future therapeutic development.

Home-monitoring of specific branched chain amino acids

Awardee: Lital Alfonta

Institution: Ben-Gurion University of the Negev

Grant Amount: $71,985.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

The project aims at developing a non-invasive home test for immediate monitoring of the orphan condition Maple Syrup Urine Disease (MSUD). MSUD is a serious genetic condition caused by a deficiency or a mutation in the enzyme keto-acid-dehydrogenase. Blood tests show high levels of three amino acids (leucine, isoleucine and valine), that can lead to life-threatening cerebral oedema and dysmyelination in affected individuals. The disease is more common in Ashkenazi Jews, and in the Bedouin population in the South region of Israel. Children with MSUD must remain on a special diet that restricts the consumption of those three amino acids. Currently, the amino acid levels of children with MSUD are monitored weekly at the hospital, and the results arrive only after several days. We propose to develop a technology based on bioelectrochemistry, which combines optimization of protein function with synthetic biology tools in order to create accurate and specific sensors for immediate monitoring of the relevant amino acids. This will enable real-time diet recommendations and forgo the need for a weekly hospital visit. In addition, neonates are routinely scanned for amino acid levels, and the novel technology can supply immediate results. Immediate monitoring of amino acids is especially crucial in the developmental phase of people (i.e. children and neonates).

Development of neuroprotective gene therapy for MSUD

Awardee: John R Counsell

Institution: UCL

Grant Amount: $71,985.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

We are seeking to develop a treatment for a severe metabolic disease called Maple Syrup Urine Disease (MSUD) that results from an inability to break down substances that most of us consume daily, when following a normal diet. MSUD patients are at risk of life-threatening brain damage unless they have their diet carefully managed from birth. Even then, patients remain at risk of brain damage during periods of illness, normally due triggered by infections. At University College London, we are developing a potential cure for MSUD using a technique called gene therapy, whereby a set of genes that are defective in MSUD can be restored in relevant parts of the body. However, it is not currently known whether gene therapy needs to be targeted to the brain as part of a curative strategy. Therefore, our research team aim to investigate this, using a mouse model of the disease, with our hypothesis being that brain cells will need to be targeted in order to fully restore healthy brain development during in early childhood.

The TGFβ pathway as a therapeutic target for collagen VI-related muscular dystrophy

Awardee: Payam Mohassel

Institution: Johns Hopkins University

Grant Amount: $113,008.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Mutations in collagen VI cause a spectrum of muscle disease ranging from severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy. Collagen VI is an integral component of the extracellular matrix. When collagen VI is not functioning properly due to mutations, skeletal muscle will develop weakness, atrophy, degeneration, and fibrosis. We have recently identified alterations in regulation of the TGFβ pathway in human muscle biopsy samples of patients with COL6-related dystrophies (COL6-RD). We have also found a similar alteration of this pathway in a new mouse model of the disease, Col6a2 knockout mice. The overall goal of this project is to help identify novel therapeutic targets in COL6-RD that engage the TGFβ pathway and to test them in the mouse model. We trust that these studies will increase our understanding of this pathway in COL6-RD and pave the way for future studies of therapeutics that target this pathway.

Preclinical assessment of potential drug candidates in Vps13b knockout mice for the treatment of Cohen syndrome

Awardee: Muhammad Ansar

Institution: University of Lausanne

Grant Amount: $100,474.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Cohen Syndrome is a rare genetic disorder caused by mutations in VPS13B gene. Patients suffer from developmental, intellectual, motor, metabolic, immunologic disorders, and visual impairments. Considering the current stage of research on Cohen syndrome and VPS13B, one of the most direct way of looking for treatment possibilities is to attempt drug repurposing of FDA-approved small pharmaceutical compounds. We recently performed a high-throughput microscopy screening assay based on the ability to revert the impaired cellular phenotype (Golgi morphology) of human VPS13B-deficient cells as well as in patients’ fibroblasts. We screened a library of 1280 FDA-approved pharmaceutical compounds, and the top 30 positive hits were further subjected to determine the dose-response. Out of the 30 positive candidates, we selected the four most effective compounds, previously used successfully in mice for the treatment of other pathologies, for which the toxicity is known, the effective dose is low, and there are little to no side effects in both mice and humans. The aim of the proposed project is to assess the treatment potential of the selected four drug candidates in the Cohen syndrome mouse model. In this project, the four selected candidate drugs will be fed to four groups of pregnant mice since the beginning of pregnancy. Drugs will be orally administered by mixing in food pellets. Pups born from these pregnancies will continue receiving the drugs and will be analysed until the age of three months. The treatment efficacy of the potential drug candidates in Vps13b knockout mice will be assessed by observing the most cardinal features of Cohen syndrome in mouse models.

IV Delivery of Ex Vivo Lentiviral Corrected CD34+ Bone Marrow Cells to Treat Systemic Disease in a Canine Model of Mucopolysaccharidosis VI

Awardee: Margret L. Casal

Institution: University of Pennsylvania

Grant Amount: $60,350.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

MPS VI causes severe skeletal disease and moderate systemic disease. Our canine model faithfully mimics the disorder in human patients with MPS VI. This projects examines the feasibility of harvesting stem cells from dogs affected with MPS VI, correcting the cells in tissue culture using viral vectors, and then returning the gene therapy corrected cells back to the affected neonatal dog. These experiments will show that this form of therapy is safe and effective, while not having the side effects of bone marrow transplantation (graft rejection, host versus graft disease, lifelong immunosuppression) or enzyme replacement therapy (repeated administration, expense, inability to correct bone disease).

Preclinical study of Fluoxetine efficacy in MPS-I mouse model

Awardee: NICOLINA CRISTINA SORRENTINO

Institution: FONDAZIONE TELETHON ETS-TIGEM

Grant Amount: $60,000.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Mucopolysaccharidosis type I (MPS-I) is a severe inherited disorder characterized by deficient activity of lysosomal enzyme α-L-Iduronidase (IDUA) responsible for the degradation of the glycosaminoglycans, leading to systemic symptoms and a shortened lifespan. Current therapies are mainly palliative with no benefit for the brain pathology. Several works indicated the importance of the lysosomal and autophagy alterations as major players in the development of brain and peripheral tissue pathology in Lysosomal Storage Disorders (LSD). Importantly, in our recent work we combined automated microscopy screening and repurposing of FDA compounds to identify approved drugs able to correct lysosomal dysfunction in LSD. Our drug survey resulted in the identification of Fluoxetine, a central nervous system drug and one of the most prescribed medicines in adults and children. Interestingly, we showed that Fluoxetine boosts lysosomal function and promotes glycosaminoglycans degradation in MPS-IIIA, MPS-I and MSD cell lines. Furthermore, our recent preclinical study demonstrated the effectiveness of Fluoxetine in the amelioration of brain and somatic pathological hallmarks of MPS-IIIA such as the accumulation of storage materials, inflammation, and slow-down cognitive deterioration in MPS-IIIA mouse model. Based on these promising results, we propose to validate the effectiveness of the Fluoxetine administration for the treatment of brain and peripheral pathology in a mouse model of MPS-I.

Characterization of the kidney pathology in Mucolipidosis type IV (MLIV)

Awardee: Diego Luis Medina

Institution: Telethon Institute of Genetics and Medicine (TIGEM)

Grant Amount: $53,634.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

TRPML1 (Transient Receptor Potential cation channel, Mucolipin subfamily1) is a non-selective cation channel that localize on lysosomal membrane. The protein TRPML1 is encoded by MCOLN1 (mucolipin1) gene. TRPML1 is the major calcium-release channel on the lysosomal membrane. TRPML1 activity is involved in a variety of membrane trafficking processes such as lysosome to TGN (Trans-Golgi-Network) retrograde trafficking, AV-lysosome fusion, lysosome reformation, and lysosomal exocytosis. Mutations in TRPML1 cause mucolipidosis type IV (MLIV: OMIM 252650), an autosomal recessive LSD (lysosomal Storage Disease) characterized by psychomotor alteration, corneal opacities, and achlorhydria, but its role in kidney it’s not completely understood. Recently acute or chronic renal diseases been observed in a subset of patients affected by MLIV. The host laboratory has an extensive expertise in the study of the cell biology processes controlling lysosomal signaling through TRPML1. Recently, Medina’s laboratory has observed that pharmacological activation of TRPML1 can induce autophagosome biogenesis through the generation of PI3P (phosphatidylinositol 3-phosphate) via Vps34 complex (Vacuolar Protein Sorting 34), thus suggesting that alterations in Vps34 pathway might be part of the pathologic features of MLIV. interestingly, this novel pathway is impaired in human fibroblasts from MLIV patients (Scotto-Rosato et al, 2020). Autophagosome is a cellular organelle characterize by characterized by a double layer membranes. It is the key structure in macro autophagy, the intracellular degradation system for cytoplasmic contents. In this proposal our principal goal is to investigate the physiological relevance of TRPML1 in kidney using an in vivo mouse model of MLIV (mouse model available in the animal facility of our institute). Thus, in addition to general parameters such as weight and size of kidney organs, we will analyze the general morphology of nephron by using specific markers of kidney cells. Also, we will investigate kidney functionality defects including (i) inflammation and (ii) proteinuria. Its’ know that prevalence of inflammation is inversely related to the level of kidney function and positively associated with the magnitude of proteinuria. There are many factors that contributes to the inflammation status as well as increased production of proinflammatory cytokines, we will study the principal biomarkers of inflammation. The second goal will be studied by using state-of-the-art proteomics from urine samples of MLIV mice. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results in the field of nephrology. The benefits of employing urinary proteomics for biomarker discovery are that urine is readily available, easy to collect and provides a renewable and non-invasive means of monitoring a patient over time. At the cellular level, we will determine the consequences of TRPML1 depletion on (i) the endocytic pathways and (ii) autophagy. Interestingly, our preliminary data in kidneys show that MLIV mice exhibit a low molecular weigh (LMW) that suggest a defective apical recycling mediate endocytosis (ARME) in Proximal Tubule (PT), section of nephron that captures all protein that pass through the glomerulus), lysosomal mislocalization and swelling of structures labelled by LAMP-1, these data suggest a block of autophagy. Furthermore, the MLIV kidneys show a TFEB nuclear translocation, an increased signal of galectin-3 and F4/80 (major macrophage marker). Understanding the mechanism of kidney damage and renal failure in MLIV is of critical importance. It is known that inhibition of calcineurin the kidneys by CSA can lead to acute and chronic injury. We will focus on the axis: TRMPL1-TFEB-Calcineurin signaling. Since renal disease in both human and model are poorly defined, we contacted and established a scientific collaboration Dr. Albert Misko, which is running the biggest natural history study in MLIV patients, and Prof. Yulia Griskuck a world-recognized expert in the study of MLIV disease (both from the MGH, US). This collaboration will allow to share mouse samples and compare the emerging mouse renal pathology with the natural history data from patients. The knowledge from these studies might be of interest for further studies to characterize and develop novel therapeutics to treat this devastating disease.

Biochemical investigation of NUBPL disease mechanisms and therapy candidates

Awardee: Eiko Ogiso

Institution: Children's Hospital of Philadelphia

Grant Amount: $120,465.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

The goal of this project is to unveil NUBPL disease mechanisms and accelerate identification of therapeutic candidates that improves health in human NUBPL disease patients. Toward this goal, we utilize three NUBPL-/- genetic disease models, C. elegans (worm, invertebrate), D. rerio (zebrafish, vertebrate), and human patient cells. First, we will investigate therapeutic efficacy of top drug candidates recently identified in other complex I disease worms as well as nutrient and signaling pathway modulators (including vitamins and dietary supplements), in our NUBPL-/- zebrafish and human patient fibroblasts. Second, we will expand our focus on iron regulation and metabolism including response to iron therapy, which might also be involved in NUBPL disease pathogenesis outside complex I deficiency. Third, we will generate induced pluripotent stem cells (iPSCs) from NUBPL patient cells and differentiate neurons from iPSCs to develop a model to study neuronal-specific effects of NUBPL disease and validate efficacies of candidate therapies at neuronal levels.

Utilizing the PS Gene Editing System to Encode a Blood‐Brain Barrier‐Penetrating IDUA as a Treatment for Murine Hurler Syndrome

Awardee: Michael Przybilla

Institution: University of Minnesota

Award Amount: $165,000

Funding Period: February 1, 2023 - January 31, 2024

The new lysosomal activator GHF201 (144DG11) as a potential treatment for mucopolysaccharidosis I”

Awardee: Or Kakhlon

Institution: Research Fund of the Hadassah Medical Organization

Award Amount: $165,000

Funding Period: February 1, 2023 - January 31, 2024

Choroid plexus disruption in CDD

Awardee: Michela Fagiolini

Institution: Boston Children's Hospital

Grant Amount: $50,240.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

In the absence of CDKL5 we discovered a significant disruption of Choroid Plexus (ChP) organization at the level of tight junctions and polarity of selective cellular transporters in the epithelial cells. These preliminary results raise the question whether the loss of CDKL5 negatively affects the function of the blood-cerebrospinal fluid (CSF)-barrier and the composition of the CSF. The ChP-CSF system delivers important growth-active molecules throughout the brain, guaranteeing its proper maturation and functioning. Here we propose 1) a detailed characterization of the development of the ChP epithelial cells in the absence of CDKL5 and 2) a multi-omics approach to profile both CSF and ChP in CDKL5 deficient disorder (CDD) mice. Due to the confined but accessible localization of the ChP, our results may establish the ChP as a new tractable target for CDD intervention.

Developing a spatiotemporal gene atlas in iMCD lymph node tissue to identify diagnostic biomarkers and therapeutic targets

Awardee: David Fajgenbaum

Institution: University of Pennsylvania

Grant Amount: $60,570.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Human Herpesvirus (HHV)-8-negative, idiopathic multicentric Castleman disease (iMCD) is a deadly hematologic illness with an unknown etiology that affects individuals of all ages. Although the causes, key immune cell types, signaling pathways, and cytokines involved are poorly understood, a key hallmark of iMCD includes characteristic lymph node features that are used for diagnosis. Despite importance of these lymph node features as diagnostic criteria, the underlying biological mechanisms driving them are not well understood. In the proposed LOI, we will develop a single cell RNAseq/spatial gene expression atlas in iMCD lymph nodes to characterize cell subpopulations, identify cell-type compositional changes, infer intercellular communication networks, and interrogate dysregulated cell types/cell locations within disease tissue. By completing the proposed specific aims we will gain important insights into lymph node biology in iMCD and the thorough characterization of the inflamed tissue will help identify new biomarkers and uncover dysregulated cell types that may be contributing to disease pathogenesis and pathology.

Advance in PIP2 treatment to restore capillary blood flow

Awardee: Fabrice Dabertrand

Institution: University of Colorado Anschutz Medical Campus

Grant Amount: $117,734

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Neurons in the brain have few energy reserves and therefore depend on local blood flow through arterioles and capillaries for a continuous supply of nutrients. A deficit in cerebral blood flow hemodynamics is an early feature of CADASIL, which suggests that cerebrovascular dysfunction has a key role in the pathogenesis leading to dementia. We recently discovered how to rescue the coupling between neuronal activity and local blood flow regulation using a phospholipid-based treatment with PIP2. However, PIP2 can act on several targets, including as an inhibitor of TRPV4 channels, an important player in capillary blood flow regulation. The proposed work aims at characterizing the impact of CADASIL on this pathway and further develop the PIP2 treatment to improve cerebral blood flow in CADASIL mouse model.

BOOSTING AUTOPHAGY AS A THERAPEUTIC STRATEGY TO TREAT TBC1D24 EPILEPSY

Awardee: Anna Fassio

Institution: University of Genoa

Grant Amount: $103,546.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

TBC1D24 has been described by our group and others to play different roles in the brain, however it is not immediate to translate this knowledge in a therapeutic strategy to treat individuals bearing TBC1D24 mutations. We recently identified a degradative cellular process, named autophagy, to be dysfunctional in neuronal cells upon loss of TBC1D24 function. Autophagy is a multistep process responsible for the removal of superfluous or dysfunctional cell components, and it is essential to guarantee neuronal health. Autophagy can be activated by different compounds, and research on small drugs acting on this process is ongoing both in the field of tumors and neurodegenerative diseases. We hypothesize that the use of these new compounds is effective in reverting the autophagy impairment and ameliorates the epileptic phenotype in TBC1D24 patients.

Development of novel gene therapy strategies for treatment of all SCN2A Disorders

Awardee: Samuel Young

Institution: University of Iowa

Grant Amount: $61,068.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

SCN2A disorders comprise a complex landscape of both missense and protein-truncating variants, resulting in a diversity of phenotypes that include epilepsy and intellectual disability. Currently, there is no cure for SCN2A Disorders, nor are there methods in development that would provide therapeutic intervention for all forms of SCN2A Disorders. Here, our team proposes proof-of-principle studies that could be beneficial for both missense and protein-truncation cases, providing a single method to treat the entire diversity of SCN2A Disorders.

The central conducting lymphatic system in patients with the Rasopathies Noonan Syndrome and CardioFacioCutaneous Syndrome with and without lymphatic disease

Awardee: Jos M Draaisma

Institution: Radboud University Medical Center

Grant Amount: $60,100.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

To get a better insight into the central lymphatic system in adult volunteers with Noonan Syndrome (NS) and CardioFacioCutaneous (CFC) Syndrome without clinical symptoms or signs of lymphatic disease compared to healthy adult volunteers without disease and NS and CFC patients with severe lymphatic disease. This to enable therapy with MEK-inhibitors or lymphovenous anastomosis. The Dutch Noonan Syndrome Foundation participates in this study.

A novel Zebrafish model of Kaposiform Lymphangiomatosis: A tool for drug discovery

Awardee: Karina Yaniv

Institution: Sheba Medical Center

Grant Amount: $68,650.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

Kaposiform lymphangiomatosis (KLA) is a member of a broad family of complex lymphatic anomalies (CLA)- rare disorders characterized by the abnormal proliferation of lymphatic vessels in the skin and internal organs. KLA, the most aggressive and rarest form of these disorders, can occur at any age, but the incidence is highest in children and teenagers. Current pharmaceutical treatments are aimed chiefly at managing the symptoms; thus, the 5-year survival rate for children affected by KLA is only about 50%. Therefore, there is an urgent need for new pre-clinical models recapitulating the disease and enabling the identification of novel drug targets. This study aims to characterize a novel KLA zebrafish model we recently established in our lab and to screen for new avenues of treatment. Because of their small size, transparency, and large progeny, ZF have become an attractive means for assessing compound effects at early stages of drug discovery. Recently, a lifesaving treatment for a lymphatic anomaly was identified through a chemical screen based on our early establishment of the ZF as a superb model for the study of lymphatic biology. Here we will harness the power of our novel mutants to screen for compounds that selectively revert the KLA-related phenotypes. We anticipate that completion of this study will help increase our understanding of the etiology of KLA and will lead to the identification of new efficient therapies.

Implementing detection of low-level mosaic variants from blood samples in hyperinsulinism to improve diagnosis

Awardee: Sarah Flanagan

Institution: University of Exeter Medical School

Grant Amount: $70,920.00

Funding Period: February 1, 2023 - January 31, 2024

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Summary:

A genetic diagnosis improves treatment and management of hyperinsulinsm; however, currently ~40% of children referred for genetic testing do not receive a genetic diagnosis. One reason for these missed diagnoses is that some children have mosaic variants, which cannot be detected by standard DNA sequencing. Mosaic variants occur during development so will only be present in some tissues, which is why they are hard to detect in genetic testing of blood samples. Our preliminary data identified 8 patients with previously undetected mosaic variants. Our study will develop these methods for detecting mosaic variants so that they can be included in diagnostic genetic testing for hyperinsulinsm worldwide.