Pharmacological induction of autophagy to treat CHM

Awardee: Ivan Conte

Institution: University of Naples Federico II

Award Amount: $64,990

Funding Period: February 1, 2021 - January 31, 2022

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Final Report Lay Summary:

The main goal of this project was to characterize at fine scale the contribution of molecular network regulating the autophagy pathway in the Retinal Pigment Epithelial ce ls. We demonstrated that impairment of ce l clearance in the RPE may affect visual system in the Choroideremia. Interestingly, we put the basis for developing a new therapeutic strategy to treat CHM, which stil represents a cha lenge, Remarkably, the therapy described herein was safe and wel tolerated showing no adverse effects associated with pharmacological treatment up to 3 months of treatment, the longest time for which the drug was tested on CHM mice. Lately, in vitro studies using hiPSC-derived RPE ce l sheet give us the opportunity to move forward with regulatory studies prerequisite to clinical trials for choroideremia patients.

Maximising the Utilisation of the Hyperinsulinism Global Registry (HIGR) [Max HIGR]

Awardee: Indraneel Banerjee

Institution: University of Manchester, Royal Manchester Children's Hospital

Award Amount: $73,190

Funding Period: February 1, 2021 - January 31, 2022

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Summary:

The HI Global Registry (HIGR) is a unique rare disease patient registry developed by Congenital Hyperinsulinism International. This international online registry that gathers important information on different types and treatments for low sugars due to hyperinsulinism. The completion of HIGR relies on parents and families uploading their child's required details. Our proposed study "Maximizing the Utility of HIGR" (MaxHIGR) aims to build on the opportunity to add medical grade information to existing parent reported HIGR information, thereby joining up clinical and parent perspectives in the search towards better understanding and improved treatment for HI. MaxHIGR will lay the basis for HIGR to evolve into a registry that will tell us about the natural history of disease, which treatments are better and have less side effects and how we can improve the quality of life of children and families living with HI.

Final Report Summary:

MaxHIGR has brought international co laborators to agree on a common data colection to replicate natural history of Congenital Hyperinsulinism. MaxHIGR required close co laboration across time zones around the world, while adapting to a a common set of rules for data entry. The form is now currently being integrated into an online tool for use in a pilot study. This study has been delayed to accommodate in person patient-clinician consultations that had been set back due to the pandemic.

HB-EGF/EGFR signaling in capillary dysfunction in CADASIL

Awardee: Masayo Koide

Institution: University of Vermont Larner College of Medicine

Award Amount: $82,795

Funding Period: February 1, 2021 - January 31, 2022

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Summary:

CADASIL, short for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarct and Leukoencephalopathy, is the most common genetic cause of a pathology known as small vessel disease (SVD) of the brain. During SVDs such as CADASIL, the structure and function of small blood vessels (arteries, arterioles, venules, and capillaries) within the brain become compromised. An early result of this vascular dysfunction is a decrease in blood flow to the brain (cerebral blood flow or CBF), which eventually leads to dementia and/or strokes. This study will elucidate the molecular mechanisms of compromised CBF increases in response to neural activity (“functional hyperemia”) using a clinically relevant CADASIL mouse model. We will specifically focus on examining the impact of CADASIL on capillaries, the smallest and most abundant vessels in the brain, which we have previously shown to be the molecular cornerstone in functional hyperemia responses in healthy animals. Considering that it is known that CADASIL causes an abnormal accumulation of specific proteins around the outside of small vessels in the brain, we propose to examine how two of these proteins, epidermal growth factor receptor (EGFR) and heparin-binding EGF-like growth factor (HB-EGF), contribute to capillary dysfunction in CADASIL. This project, by providing a greater understanding of the cellular pathways contributing to CADASIL pathologies, will create a firm footing for future therapeutic development.

Publications:

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity

Final Report Lay Summary:

CADASIL, short for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarct and Leukoencephalopathy, is the most common genetic cause of a pathology known as smal vessel disease (SVD) of the brain. During SVDs such as CADASIL, the structure and function of smal blood vessels (arteries, arterioles, venules, and capilaries) within the brain become compromised. An early result of this vascular dysfunction is a decrease in blood flow to the brain (cerebral blood flow or CBF), which eventua ly leads to dementia and/or strokes. This study examined the molecular mechanisms of compromised CBF increases in response to neural activity (“functional hyperemia”) using a clinica ly relevant CADASIL mouse model. We specifica ly focused on examining the impact of CADASIL on capilaries, the sma lest and most abundant vessels in the brain, which we have previously shown to be the molecular cornerstone in functional hyperemia responses in healthy animals. Considering that it is known that CADASIL causes an abnormal accumulation of specific proteins around the outside of sma l vessels in the brain, we examined how two of these proteins, epidermal growth factor receptor (EGFR) and heparin-binding EGF-like growth factor (HBEGF), contribute to capilary dysfunction in CADASIL. State-of-art techniques, including laser Doppler flowmetry, patch-clamp electrophysiology, two-photon microscopy and a newly developed capilary-arteriole continuum preparation were used to examine the role of capilary EGFR signaling in causing cerebral blood flow deficits in CADASIL model mice. Notably, we found that CADASIL-induced functional hyperemia deficits were caused by insufficient phosphatidylinositol 4,5-bisphosphate (PIP2), an endogenous activator of Kir2.1 potassium channels in capilary EC membranes. Furthermore, our data demonstrates that PIP2 content in capilary EC membranes can be modified by HB-EGF/EGFR signaling. In fact, the application of PIP2 or stimulation of EGFR restored capilary Kir2.1 channel activity and functional hyperemia in CADASIL mice. These results support the concept that HB-EGF/EGFR signaling modulates PIP2 content in capilary EC membrane, EC Kir2.1 channel activity, and functional hyperemia. In summary, we demonstrated a novel mechanism underlying functional hyperemia deficits in CADASIL. Our findings suggest that capilary HB-EGF/EGFR signaling and exogenous PIP2 administration may have potential as therapeutic targets as CADASIL treatments. This project, supported by the 2021 Milion Do lar Bike Ride Pilot Grant program, provides a wealth of new information to deepen our understanding of the pathologies involved in CADASIL and other smal vessel diseases of the brain and creates a foundation for future therapeutic studies.

Development of at-home speech and wearable sensor biomarkers for ataxia-telangiectasia

Awardee: Anoopum Gupta

Institution: Massachusetts General Hospital and Harvard Medical School

Award Amount: $129,898

Funding Period: February 1, 2021 - January 31, 2022

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Final Report Lay Summary:

Promising disease-modifying therapies are being developed for ataxia-telangiectasia and other pediatric neurological diseases, but current assessment tools are very insensitive at determining efficacy, resulting in large and expensive trials. This project aimed to develop precise motor outcome measures, using inexpensive and widely accessible digital technologies, that can sensitively determine if a therapy is effective in children of a l ages. We co lected continuous wrist accelerometer data from 31 individuals with ataxiatelangiectasia and 27 controls aged 2-20 years old. Longitudinal wrist sensor data were colected in 14 ataxia-telangiectasia participants and 13 controls. A novel algorithm was developed to extract wrist movement patterns the accelerometer data. Wrist sensor features were compared with caregiver-reported motor function and ataxia severity on neurologist-performed ataxia rating scales. We found that these wrist sensor-based features show strong potential as novel disease measures for clinical trials: they demonstrate high reliability, correlate with clinician assessments of motor severity and caregiverreported motor function, and show potential to sensitively quantify disease progression. By passively measuring everyday activity, the information obtained can be more ecologica ly valid and comprehensive than task-specific measurements and is applicable in young children as we l in older, non-ambulatory individuals.

Characterization and correction of Cdkl5 nuclear function in human CDD neurons during maturation

Awardee: Simone DiGiovanni, MD, PhD

Institution: Imperial College London

Award Amount: $149,277

Elucidate the neural mechanisms underlying sleep disturbances and the relation between seizures and sleep-wake cycle in CDKL5 deficiency disorder (CDD)

Awardee: Liqin Cao, PhD

Institution: Tsukuba University

Award Amount: $139,940

Visualizing the effect of CDKL5 deficiency on ciliary core structures

Awardee: Alan Brown, PhD

Institution: Harvard Medical School

Award Amount: $150,000

Incorporation of functional unnatural amino acids into CDKL5 to study its function in cells

Awardee: Maolin Guo, PhD

Institution: University of Massachusetts, Dartmouth

Award Amount: $150,000

Ascertaining the cell-autonomous effects of CDKL5 mutations in every brain cell type

Awardee: Steven A. McCarroll, PhD

Institution: Harvard Medical School

Award Amount: $140,000

Assessing peripheral sensory neuron dysfunction in animal models for CDKL5 deficiency disorder

Awardee: Lauren Orefice, PhD

Institution: Massachusetts General Hospital

Award Amount: $150,000

CDKL5 regulation of ciliary assembly and signaling

Awardee: Gregory J. Pazour, PhD

Institution: University of Massachusetts Medical School

Award Amount: $150,000

Structural/functional characterization of full-length hCDKL5 isoform 1 produced in recombinant marine bacteria and use of pharmacological chaperones to stabilize hCDKL5 missense mutants

Awardee: Maria Luisa Tutino, PhD

Institution: University of Naples

Award Amount: $148,300

A working prototype of an in-home ionized calcium monitoring device using a paper-based ion-selective optode and an optical reader

Awardee: Xuewei Wang, PhD

Institution: Virginia Commonwealth University

Award Amount: $149,019

Funding Period: January 1, 2020 - December 31, 2020

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Project Summary:

We are developing test strips for ionized calcium in finger-prick blood samples. One drop of blood can be easily introduced into the strip by patients. The optical response of the strip is recorded by a regular smartphone equipped with a customized app. The test can be finished within two minutes because of the fast sensor response. The concentration of ionized calcium can be accurately determined in a range of 0.1 to 5.0 mmol/L (0.4 to 20.0 mg/dL). There is no interference from other molecules and ions in the blood. Therefore, this new technology will enable the in-home measurement of calcium in the blood and allows the management of hypoparathyroidism by the patient themselves.

Final Summary:

Affordable and portable blood calcium sensors using a smartphone detector have been developed. These sensors empower patients to measure their calcium ion concentration at home using blood collected by fingerstick.

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Publications:

R. Wang, X. Wang. Sensing of inorganic ions in microfluidic devices. Sensors and Actuators B: Chemical 2021, 329, 129171

R. Wang, Y. Zhou, N. Ghanbari Ghalehjoughi, Y. Mawaldi, X. Wang. Ion-Induced Phase Transfer of Cationic Dyes for Fluorescence-Based Electrolyte Sensing in Droplet Microfluidics. Analytical Chemistry, 2021

N. Ghanbari Ghalehjoughi, R. Wang, S. Kelley, X. Wang. Ultrasensitive Ionophore-Based Liquid Sensors for Colorimetric Ion Measurements in Blood. Analytical Chemistry, 2023, 95, 12564-12564

Developing human pluripotent stem cells for investigation and treatment of hypoparathyroidism

Awardee: Rene Maehr, PhD

Institution: Umass Medical School

Award Amount: $500,000

Funding Period: January 1, 2020 -December 31, 2020

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Summary:

The parathyroid gland is critically involved in regulation of calcium homeostasis of the body. Hypoparathyroidism as encountered by parathyroid damage, hypoplasia, or as a result of thyroid and parathyroid surgery, results in chronic hypocalcemia and low-turnover bone disease. Human pluripotent stem cells could provide a virtually unlimited source of parathyroid-like cells with calcium level responsiveness, offering a unique opportunity for development of a cell replacement products capable of regulating calcium levels. To unlock human pluripotent stem cell-based treatment strategies, robust and safe stem cell differentiation protocols need to be established. Here, we propose to develop an approach that is based on human pluripotent stem cell differentiation according to a developmental roadmap, and cutting edge humanized mouse avatar models for functional evaluation of human parathyroid-like cells. We expect this rigorous approach to provide several high-impact resources, including a source of high-fidelity human parathyroid-like cells and novel mouse models for studying parathyroid function.

Publication:

Integration of single-cell transcriptomes and chromatin landscapes reveals regulatory programs driving pharyngeal organ development - Nature Communitcations

In vitro differentiation of parathyroid cells from stem cells

Awardee: Michael Mannstadt, MD

Institution: Massachusetts General Hospital/Harvard University

Award Amount: $1,000,000

Funding Period: January 1, 2020 - December 31, 2021

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Summary:

Parathyroid glands produce parathyroid hormone (PTH), which is necessary for regulating blood calcium and phosphate levels and maintaining bone health. Patients with insufficient parathyroid gland activity (hypoparathyroidism) can suffer from multiple symptoms caused by low blood calcium levels, including minor problems like muscle twitching or severe, life-threatening complications such as tetany and seizures. Conventional treatment with calcium and active vitamin D does not replace the functions of PTH and can lead to undesired long-term effects, such as kidney stones. PTH replacement therapy requires daily self-injections. 

Currently, testing of serum calcium involves a visit to a clinical laboratory, a blood draw, and a delay while the patient waits for a report of their test results. This delays dose adjustment and leads to hyper- or hypocalcemia.

The long-term goal of this proposal is to offer a regenerative therapy for patients with hypoparathyroidism using mature parathyroid cells differentiated from human stem cells.  With our collaborators from several institutions, including stem cell and developmental biologists, parathyroid surgeons, and specialists in microencapsulation of human stem cell-derived hormone-producing cells, we aim to define genetic mechanisms governing parathyroid cell fate specification during embryonic development.  We will target critical pathways using small molecule activators and inhibitors to facilitate parathyroid cell fate specification.  We will also test a novel microencapsulation technique for human parathyroid cells by transplantation in mice.