Our multidisciplinary team aims to uncover how PRDM1 and pioneer transcription factors (TFs) work together to reprogram human fibroblasts into liver cells. Using a unique method that transduces a pool of barcoded TFs and tracks their activities in individual cells using single-cell multiome assays, we can understand how they control genes across the entire genome. This research will help us improve how we direct cell fate, which is crucial for better modeling pediatric diseases, as well as advancing regenerative medicine.

Awardees: Makiko Iwafuchi, PhD, Hee-Woong Lim, PhD, Emily Miraldi, PhD

Amyotrophic lateral sclerosis (ALS) is a complex disease affecting nerve cells in the brain and spinal cord, leading to muscle weakness and paralysis. About 10-15% of ALS cases are inherited due to genetic mutations. In a large family with ALS caused by a specific mutation in the Fused in Sarcoma (FUS) gene, some individuals carrying this mutation did not develop ALS even into their 60s. This suggests that certain protective genes may prevent the development of ALS. We identified a rare genetic variant that might offer protection against ALS. To understand how this protective gene works, we will use stem cells derived from patients with ALS and their motor neurons, grown in the lab. By comparing cells with and without this genetic variant, we aim to uncover the molecular reasons behind this protective effect against ALS.

Awardee: Ziyuan Guo, PhD

Fragile X is the most common inherited single-gene cause of autism and intellectual disabilities, and is caused by a cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the FMR1 gene located on the X chromosome. The disease severity varies substantially among patients, some are nonverbal and others obtain college degrees; however, there are no quantitative predictors of disease severity or outcomes. This study will use novel long-read sequencing technology to more precisely determine CGG repeat length and methylation and evaluate its ability to predict disease severity in FXS. Better defining the FXS genotype will improve diagnostics and may help predict disease severity and outcomes in order to optimize individualized treatment strategies.

Awardees: Lauren Schmitt, PhD, Christina Gross, PhD, Craig Erickson, MD

Pathogenic variants in homeodomain genes such as ARX, PHOX2B, SHOX, and ALX4 are associated with many congenital birth defects and human diseases. How most homeodomain variants impact function and cause disease is not well understood. The goals of this collaborative grant are twofold: First, we will develop quantitative high-throughput assays to define how homeodomain variants associated with disease impact DNA binding. Second, we will use human forebrain organoids and genomic assays to identify the key downstream target genes of ARX, a disease gene associated with congenital brain malformations, intellectual disability, epilepsy, and autism.

Awardees: Brian Gebelein, PhD, Jason Tchieu, PhD

Critical limb ischemia is a major public health crisis with escalating prevalence. We recently found a highly proangiogenic mesenchymal progenitor population within human subcutaneous adipose tissue that express the surface receptor CD271. Transplantation of these progenitors into ischemic limbs in vivo effectively reverses limb ischemia and facilitates limb salvage. However, the fundamental molecular mechanisms underpinning the pro-angiogenic effects induced by CD271 signaling remain unknown. In this application, we will elucidate the signaling pathways and the transcriptional regulatory networks that orchestrate the pro-angiogenic activity induced by CD271 signaling.

Awardees: Juan Sanchez Gurmaches, PhD, Hee Woong Lim, PhD

New cellular and immunotherapeutic strategies are desperately needed to eliminate malignant clones and halt progression of cancers with poor treatment outcomes. In this CpG grant we will build a pancancer atlas of splicing neoantigens to nominate, rank, and compare novel targets for the creation of new cancer therapies. These analyses are expected to reveal thousands of highly shared splicing neoantigens, derived from distinct regulatory mechanisms, that are both specific to and recurrent across malignancies. The proposed work utilizes breakthrough methods in the detection and interactive exploration of splicing neoantigens.

Awardees: Nathan Salomonis, PhD, Susanne Wells, PhD, Daniel Schnell, PhD, H. Leighton Grimes, PhD

Juvenile Idiopathic Arthritis (JIA) is the most common autoimmune disease in children that can result in lifelong disability. Inflammation and immune cell infiltration in the synovium drives joint destruction and progression, however, no studies to date have robustly examined this tissue in JIA to find factors linked to disease. In this study, we will leverage cutting-edge technologies to identify cellular features associated with disease progression in order to discover new avenues for therapeutic treatment and biomarkers of disease progression.

Awardees: Yuriy Baglaenko, PhD, Patricia Vega-Fernandez, MD, MSc, RhMSUS, Grant Schulert, MD, PhD, Sara Szabo, MD, PhD, Yunguan Wang, PhD

RASopathies are a group of developmental disorders that exhibit a variety of abnormalities including neurological defects. The cell specific impact and underlying cause of neurological defects remains an active area of research in the field. In this CPG pilot application, we will investigate the relationship between RASopathy mutations and oxidative cell stress pathway activation in the mouse model system. If successful, the CPG pilot award will provide key genetic tools and gene expression data to better understand how RASopathy or neurodevelopmental mutations effect distinct brain cells.

Awardee: Ronald Waclaw, PhD

Inflammatory Bowel Disease (IBD) is a chronic and often debilitating autoinflammatory condition that impacts nearly 3 million Americans, with an increasing incidence in children world-wide. Current therapies lead to sustained healing in fewer than one-third of patients, highlighting the need for effective alternatives. Here we use cutting-edge technologies (single-cell genomics, mathematical modeling, human organoids) to improve patient-specific tissue models of IBD, for design and screening of therapeutic strategies.

Awardees: Emily Miraldi, PhD, Lee A. Denson, MD

Primary sclerosing cholangitis (PSC) is a rare liver disease where the bile ducts get inflamed and scarred over time. Current treatments don't effectively target this scarring, so we need new approaches. Our previous research suggests that a certain type of immune cell, called M2 macrophages, might be important in causing this scarring by communicating with cells around the bile ducts. We plan to employ the new spatially resolved transcriptomics technology to study samples from healthy people and those with different stages of PSC to understand how these cells interact and cause scarring. If successful, this could lead to new treatments to control the progression of PSC.

Awardees: Yunguan Wang, PhD, Alexander Miethke, MD

Heterozygous mutations in the KMT2D gene cause Kabuki Syndrome type 1 (KS1), a rare multi-organ disease where individuals suffer from developmental defects, including congenital heart defects, facial dysmorphism and intellectual disability. Here, we will use induced pluripotent cell system to understand the neurodifferentiation defects associated with KMT2D haploinsufficiency. This pilot project will provide a solid foundation for the study of the mechanism(s) behind neurodevelopmental defects in KS1 and allow identification of molecular targets for potential therapies.

Awardees: Artem Barski, PhD, Jason Tchieu, PhD & Sarah Potter, PhD

Cholestatic liver diseases are characterized by failures of draining bile from the liver and represent the majority of liver diseases in children. About 30% of children with cholestasis do not have a molecular diagnosis, hindering effective treatment. We previously associated rare deleterious variants in ABCC12, which encodes a novel transporter MRP9, with intrahepatic cholestasis. In this application, we will investigate the roles of MRP9 in cholangiocyte health and test if MRP9 deficiency is a novel risk factor in biliary fibrosis, the common final pathway of cholestatic liver diseases in children.

Awardees: Chunyue Yin, PhD & Alexander Miethke, MD

Mutations in the GNAQ gene are a cause of childhood vascular tumors. These vascular tumors are very disfiguring and can be life-threatening. However, there are no FDA-approved treatments available that specifically target tumor biology. Using a new genetic mouse model and adeno-associated viruses (AAVs) that preferentially infect tumor-forming endothelial cells in blood vessels, the Boscolo and Robinson labs in the Division of Experimental Hematology and Cancer Biology will design and test a novel targeted gene therapy for GNAQ-driven vascular tumors in GNAQ mutant mice. If successful, this CpG Pilot Award will provide proof-of-concept results that are needed to support further development of AAV-based treatments for GNAQ-mediated vascular tumors.

Awardees: J. Elliott Robinson, MD, PhD, Elisa Boscolo, PhD

Mechanical circulation (heart-lung machines), which includes extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass (CPB), are lifesaving devices that provide support for patients with heart and lung failure, as well as patients undergoing complex open-heart surgery. Unfortunately, ECMO and CPB can be complicated by serious blood clots and immune system dysfunction, both of which can cause life-threatening organ damage. Our studies in animal models indicate that these clotting and immune side effects are linked through a clotting system protein called factor XII. Unlike other proteins in the clotting system, we can eliminate factor XII without creating any bleeding risks, making it a potentially exciting target for new therapies. The studies in our CpG grant will use cutting-edge genetic technologies to define the effects of factor XII and mechanical circulation on individual immune cell subtypes in molecular detail, providing critically needed information regarding how mechanical circulation and factor XII alter immune functions. We are excited by the potential of the studies funded by this grant to lead to new ways to treat and/or prevent the dangerous clotting and immune sequalae associated with ECMO and CPB.

Awardees: Joseph S. Palumbo, MD, David L.S. Morales, MD, Mousa Kharnaf, MD (not pictured) & Bruce J. Aronow, PhD

Food allergy is a potentially life-threatening condition. Current diagnostic tests either have poor accuracy or carry considerable risk and cost. We plan to address this unmet need by developing novel diagnostic biomarkers by interrogates the cell types responsible for producing this inappropriate food reactivity.

Awardees: Krishna M. Roskin, PhD, Sandra Andorf, PhD & Amal Assa’ad, MD

Genetic variants in the human SOX10 gene cause serious congenital syndromes affecting pigmentation, digestion, hearing, and neural function. This pilot project will trace the source of an additional unexpected symptom found in a new sox10 animal model: severely deficient bone mineralization associated with disrupted systemic mineral regulation. Findings will have relevance for patients with SOX10 variants, as they may harbor subclinical symptoms that could be targeted to improve clinical outcomes.

Awardee: Lindsey Barske, PhD

Pioneer transcription factors (TFs) play a crucial role in regulating cell fate by establishing transcriptionally active states. However, cell fate control requires the coordination of both gene activation and repression, a process that is poorly understood. To fill this gap, we will systematically determine the gene regulatory principle of pioneer TFs and repressors cooperating on thousands of cis-regulatory sequences, utilizing perturbation-based Massively Parallel Reporter Assays and DiMeLo-seq (Directed Methylation with Long-read-seq for mapping protein-DNA interaction). Our studies will ultimately enhance precise manipulations of cell fate in regenerative medicine.

Awardees: Makiko Iwafuchi, PhD, Hee Woong Lim, PhD & Kohta Ikegami, PhD

Genetic screens are performed for many diseases, but their power is dampened by frequent findings of alterations that have unknown effects on the function of the encoded protein; these are termed variants of uncertain significance (VUS). Here we focus on ataxia-telangiectasia (A-T), a devastating disease that typically afflicts children and which often targets the nervous system and increases the risk of developing cancer. A-T is associated with changes in the ATM gene. VUS diminish the capacity of screens of the ATM gene to aid early diagnosis and the implementation of appropriate treatments. Thus, in this CpG pilot project, we will establish a high throughput system for in vitro tests to help interpret the clinical impact of ATM VUS, thereby benefiting A-T patients and their families.

Awardees: Paul R. Andreassen, PhD, Leah Kottyan, PhD & Adam Lane, PhD