Regional Variation in Cardiovascular Genes Enables a Tractable Genome Editing Strategy.

BACKGROUND: To realize the potential of genome engineering therapeutics, tractable strategies must be identified that balance personalized therapy with the need for off-the-shelf availability. We hypothesized that regional clustering of pathogenic variants can inform the design of rational prime editing therapeutics to treat the majority of genetic cardiovascular diseases with a limited number of reagents. METHODS: We collated 2435 high-confidence pathogenic/likely pathogenic (P/LP) variants in 82 cardiovascular disease genes from ClinVar. We assessed the regional density of these variants by defining a regional clustering index. We then combined a highly active base editor with prime editing to demonstrate the feasibility of a P/LP hotspot-directed genome engineering therapeutic strategy in vitro. RESULTS: P/LP variants in cardiovascular disease genes display higher regional density than rare variants found in the general population. P/LP missense variants displayed higher average regional density than P/LP truncating variants. Following hypermutagenesis at a pathogenic hotspot, mean prime editing efficiency across introduced variants was 57±27%. CONCLUSIONS: Designing therapeutics that target pathogenic hotspots will not only address known missense P/LP variants but also novel P/LP variants identified in these hotspots as well. Moreover, the clustering of P/LP missense rather than truncating variants in these hotspots suggests that prime editing technology is particularly valuable for dominant negative disease. Although prime editing technology in relation to cardiac health continues to improve, this study presents an approach to targeting the most impactful regions of the genome for inherited cardiovascular disease.

A comprehensive, multidisciplinary, precision medicine approach to discover effective therapy for an undiagnosed, progressive, fibroinflammatory disease.

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient's serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient's cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders.

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.