"Mechanisms of transcriptional regulation by Tbx1"

Deletion 22q11.2 syndrome (22q11DS) is the most common microdeletion syndrome in man, with an incidence of approximately 1:4000 live births (1); the major malformations include congenital heart defects such as truncus arteriosus (TA) and interrupted aortic arch type B (IAA-B), hypo/aplasia of the parathyroid and thymus glands, and craniofacial dysmorphism. Velo-cardio-facial (VCFS) and DiGeorge syndromes (DGS) are other diagnoses commonly made in affected individuals (1). The gene encoding the T-box transcription factor Tbx1, which is required for pharyngeal and cardiovascular development, has been identified as the gene haploinsufficient in mouse and human. We and others have identified a number of genes potentially targeted by Tbx1, but the mechanisms by which it can regulate the transcription of these genes and how it controls developmental pathways, are moslty unclear. One of the best studied molecular functions of Tbx1 is in heart development, where it is required to sustain proliferation of mesodermally-derived precursors of the second heart field (SHF), a cardiac progenitor cell population that contributes to the development of most of the heart, including the outflow tract and right ventricle. To better understand how it works during embryonic development, we evaluated Tbx1-dosage dependent gene expression changes in vivo using a novel dosage gradient approach. Among genes sensitive to Tbx1 level, we found the one encoding the cardiogenic transcription factor Mef2c which is involved in cardiomyocyte differentiation. Interestingly, this gene was anti-correlated to Tbx1 dosage; in situ hybridization on mutant mouse embryos also corroborated quantitative expression data. These results suggest that Tbx1 may negatively regulate cardiac muscle cell differentiation through a mechanism involving Mef2c transcriptional repression; this would be consistent with recent data showing that loss of function of Tbx1 is associated with increased expression of differentiation markers of the myocardium. It has also been shown that Mef2c is a direct transcriptional target of Gata4 in the SHF, during mouse embryonic development (2); accordingly our in vitro data, suggest that Tbx1 could negatively regulate Mef2c expression, somehow interfering with Gata4-dependent Mef2c activation. Virtually all the mechanistic data obtained so far derive from murine models of 22q11DS; there is the need of a system to validate these data on human material. Since human Embryonic Stem cells can differentiate in vitro, into multiple somatic tissues, including cardiac progenitors, we generated DiGeorge syndrome-specific Pluripotent Stem cells by reprogramming adult patient fibroblasts. Developing of this system as human model of the disease, will help us to investigate its underlying molecular mechanism on a cellular level. Tbx1 loss of function in mice, and, to a lesser extent, TBX1 haploinsufficency in DiGeorge syndrome patients, is associated with hypoplasia or aplasia of several organs and tissues; so it is possible that Tbx1 function in regulating the balance between proliferation and differentiation in the SHF, may also apply to other tissues where Tbx1 is expressed. Availability of DiGeorge syndrome-specific Pluripotent Stem cells, will help us to speculate whether that disregulation of the balance between proliferation and differentiation of different types of progenitor cells or stem cells, may be a basic pathogenic mechanism for the loss of function phenotype

Tbx1 is a negative modulator of Mef2c

The developmental role of the T-box transcription factor Tbx1 is exquisitely dosage-sensitive. In this study, we performed a microarray-based transcriptome analysis of E9.5 embryo tissues across a previously generated Tbx1 mouse allelic series. This analysis identified several genes whose expression was affected by Tbx1 dosage. Interestingly, we found that the expression of the gene encoding the cardiogenic transcription factor Mef2c was negatively correlated to Tbx1 dosage. In vivo data revealed Mef2c up-regulation in the second heart field (SHF) of Tbx1 null mutant embryos compared with wild-type littermates at E9.5. Conversely, Mef2c expression was decreased in the SHF and in somites of Tbx1 gain-of-function mutants. These results are consistent with the described role of Tbx1 in suppressing cardiac progenitor cell differentiation and indicate also a negative effect of Tbx1 on Mef2c during skeletal muscle differentiation. We show that Tbx1 occupies conserved regulatory regions of the Mef2c locus, suggesting a direct effect on Mef2c transcription. However, we also show that Tbx1 interferes with the Gata4→ Mef2c regulatory pathway. Overall, our study uncovered a target of Tbx1 with critical developmental roles, so highlighting the power of the dosage gradient approach that we used

Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation canbe restored by exon skipping in both patient cardiomyocytes invitro and mouse heart invivo, indicating RNA-based strategies as a potential treatment option for DCM

Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer

The TBX1 gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription, and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here, we show that increased dosage of Tbx1 correlates with downregulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro-differentiated human induced pluripotent stem cells (hiPSCs) expressed higher levels of MEF2C and showed increased AHF acetylation, compared with hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c AHF enhancer by inducing histone deacetylation

BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

Abstract Background Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events. Results We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties. Conclusions Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors

Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes

AIMS: Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias. METHODS AND RESULTS: We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype-specific dru

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy

<div><p>Aims</p><p>Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome—decreased ventricular function. Titin (<i>TTN</i>)—a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus <i>TTN</i> mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS).</p><p>Methods and results</p><p>iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca²⁺]_out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells.</p><p>Conclusions</p><p>These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.</p></div

In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model

Abstract Background Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. Results To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. Conclusions Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia