AAV9-mediated MYBPC3 gene therapy with optimized expression cassette enhances cardiac function and survival in MYBPC3 cardiomyopathy models.

Hypertrophic cardiomyopathy (HCM) affects approximately 600,000 people in the United States. Loss-of-function mutations in Myosin Binding Protein C3, MYBPC3, are the most common genetic cause of HCM, with the majority of mutations resulting in haploinsufficiency. To restore cardiac MYBPC3, we use an adeno-associated virus (AAV9) vector and engineer an optimized expression cassette with a minimal promoter and cis-regulatory elements (TN-201) to enhance packaging efficiency and cardiomyocyte expression.

Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells.

An ideal tool for the study of cellular biology would enable the measure of molecular activity nondestructively within living cells. Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells but have historically been limited by a necessary tradeoff between spatiotemporal resolution and throughput. Here we address these limitations using oblique line scan (OLS), a robust single-objective light-sheet-based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a large field of view.

A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms.

The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis.

WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis.

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood.

AAV9:PKP2 improves heart function and survival in a Pkp2-deficient mouse model of arrhythmogenic right ventricular cardiomyopathy.

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need. Mutations in Plakophilin-2 (PKP2), encoding a desmosomal protein, account for approximately 40% of ARVC cases and result in reduced gene expression.

Functional analysis of a common BAG3 allele associated with protection from heart failure.

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism remains elusive. In this study, we used induced pluripotent stem cells to test if the only coding variant in this allele block, BAG3, alters protein and cellular function in human cardiomyocytes. Quantitative protein interaction analysis identified changes in BAG3 protein partners specific to cardiomyocytes.

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs.

Phenotypic screening with deep learning identifies HDAC6 inhibitors as cardioprotective in a BAG3 mouse model of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM.

ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain.

OCT4 and SOX2 confer pluripotency by recruiting coactivators to activate stem cell–specific transcription. However, the composition of coactivator complexes and their roles in maintaining stem cell fidelity remain unclear. Here, we report the ATP-binding cassette subfamily F member 1 (ABCF1) as a coactivator for OCT4/SOX2 critical for stem cell self-renewal.

Deep learning detects cardiotoxicity in a high-content screen with induced pluripotent stem cell-derived cardiomyocytes.

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to detect drug-induced toxicity in vitro. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

Determining cellular CTCF and cohesin abundances to constrain 3D genome models.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al.

Regulation of DNA demethylation by the XPC DNA repair complex in somatic and pluripotent stem cells.

Faithful resetting of the epigenetic memory of a somatic cell to a pluripotent state during cellular reprogramming requires DNA methylation to silence somatic gene expression and dynamic DNA demethylation to activate pluripotency gene transcription. The removal of methylated cytosines requires the base excision repair enzyme TDG, but the mechanism by which TDG-dependent DNA demethylation occurs in a rapid and site-specific manner remains unclear. Here we show that the XPC DNA repair complex is a potent accelerator of global and locus-specific DNA demethylation in somatic and pluripotent stem cells.

The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells.

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells.

Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis.

Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts).

miR-34 miRNAs provide a barrier for somatic cell reprogramming.

Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming.