Biology subjects

Freeman, Z. T.

Publications and source records attributed to Freeman, Z. T..

4 recordsLinked to original sources

bioRxiv2026

Mapping and rewiring the MYBPC3 promoter for rescue of haploinsufficiency driven hypertrophic cardiomyopathy

Autosomal dominant loss-of-function variants in the gene MYBPC3 are, collectively, the most common genetic cause of hypertrophic cardiomyopathy (HCM) and are a prototype of haploinsufficient human disease. Typical for haploinsufficiency-associated genes, hundreds of unique loss-of-function pathogenic variants have been reported for MYBPC3 - therapeutic gene editing to correct each of these variants poses major regulatory and logistical hurdles. Upregulating wild-type allele expression could offer a generalizable therapeutic strategy, but the capacity to modulate native MYBPC3 transcription is unknown. Here, we present a variant-agnostic approach to rescue haploinsufficiency by mapping and rationally redesigning the MYBPC3 promoter. Using massively parallel reporter assays (MPRAs) in human induced pluripotent stem cell-derived cardiomyocytes, we performed saturation mutagenesis of the MYBPC3 promoter at single base-pair resolution. This defined a new class of clinically relevant noncoding loss-of-function variants while revealing essential cis-regulatory grammar anchored by key transcription factor binding sites (TFBSs). Furthermore, by systematically screening thousands of variant combinations, modular promoter elements, and heterologous TFBS insertions, we identified synergistic sequence edits that drive robust increases in MYBPC3 expression. Together, our findings improve the clinical interpretation of noncoding variants and establish a scalable blueprint for promoter editing to treat MYBPC3-associated HCM and other haploinsufficient diseases.

molecular biology↗

bioRxiv2026

Genomic Engineering of Gene Dosage: A Generalizable Framework for Modeling Haploinsufficiency-Mediated Human Disorders through Splicing Modulation

Heterozygous loss-of-function variants causing gene dosage reduction underlie many human genetic disorders, yet preclinical mouse models frequently fail to recapitulate human disease phenotypes due to post-translational compensation. Here, we present a generalizable framework to modulate gene dosage by alternative splicing via genome editing. By shifting proportions of transcripts toward nonsense-mediated decay, this approach enables precise titration of functional protein levels. Applying this concept to Mybpc3, we combined hypomorphic splice-altering alleles to overwhelm post-translational buffering in mice, successfully reproducing hallmark structural and functional features of human hypertrophic cardiomyopathy. Extending this concept, we engineered a portable Modulated Alternative Splice Cassette (MASC) inserted into Dsp, yielding patient-level protein reductions and characteristic arrhythmogenic cardiomyopathy pathologies, including subepicardial cardiac fibrosis and immune cell infiltration. These novel haploinsufficient models of Mybpc3 hypertrophic cardiomyopathy and Dsp arrhythmogenic cardiomyopathy will critically enable mechanistic and therapeutic testing under protein stoichiometry conditions that closely replicate the patient disorders. Finally, systematic in silico predictive modeling across 645 human haploinsufficiency-associated genes demonstrated the generalizability of MASC insertion effects on splicing across diverse tissue types. Notably, the framework developed here leaves endogenous gene expression regulatory logic intact, allowing these models to be used to develop and validate therapeutic approaches that target transcription. Overall, this strategy provides a scalable roadmap for engineering high-fidelity animal models for human diseases caused by haploinsufficiency.

genetics↗

bioRxiv2024

The C2 domain augments Ras GTPase Activating Protein catalytic activity

Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins.

biochemistry↗

bioRxiv2023

Taxonomical and ontological analysis of verified natural and laboratory human coronavirus hosts

To fully understand COVID-19, it is critical to identify and analyze all the possible hosts of SARS-CoV-2 (the pathogen of COVID-19) and compare them with the hosts of other human coronaviruses. In this study, we collected, annotated, and performed taxonomical and ontological analysis of all the reported and verified hosts for all human coronaviruses including SARS-CoV, MERS-CoV, SARS-CoV-2, and four others that cause the common cold. A total of 37 natural hosts and 19 laboratory animal hosts of host human coronaviruses were identified based on experimental or clinical evidence. Our taxonomical ontology-based analysis found that all the verified susceptible natural and laboratory animals belong to therian mammals. Specifically, these 37 natural therian hosts include one wildlife marsupial mammal (i.e., Didelphis virginiana) and 36 Eutheria mammals (a.k.a. placental mammals). The 19 laboratory animal hosts are also classified as placental mammals. While several non-therian animals (including snake, housefly, zebrafish) were reported to be likely SARS-CoV-2 hosts, our analysis excluded them due to the lack of convincing evidence. Genetically modified mouse models with human Angiotensin-converting enzyme 2 (ACE2) or dipeptidyl peptidase-4 (DPP4) protein were more susceptible to virulent human coronaviruses with clear symptoms. Coronaviruses often became more virulent and adaptive in the mouse hosts after a series of viral passages in the mice. To support knowledge standardization and analysis, we have also represented the annotated host knowledge in the Coronavirus Infectious Disease Ontology (CIDO) and provided ways to automatically query the knowledge.

bioinformatics↗