Citations

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Introduction: Elastin-like polypeptides (ELP) are novel drug delivery vectors that can be fused to therapeutic agents and cell penetrating peptides (CPPs), slowing their plasma clearance and increasing uptake by target tissues. We developed a CPP-ELP fusion to a specific inhibitor of the p50 subunit of NF-kB (SynB1-ELP-p50i). We developed a swine model of chronic kidney disease (CKD) that also fosters hypertension, cardiac remodeling, and diastolic dysfunction, and observed that cardiac abnormalities associate with increased cardiac expression of NF-kB, a ubiquitous transcription factor that modulates cardiac inflammation, fibrosis, and vascular remodeling. Hypothesis: We aimed to define the feasibility and efficacy of intra-coronary (IC) SynB1-ELP-p50i administration to test whether a targeted inhibition of NF-kB can improve cardiac abnormalities in CKD. Methods: We first performed a biodistribution study (BDS) in 3 pigs, 4 hours after IC injection of fluorescently labeled SynB1-ELP-p50i (10 mg/kg). Then, CKD was induced in 6 pigs (bilateral renovascular disease and dyslipidemia), treated with a single IC SynB1-ELP-p50i or placebo at 6 weeks and observed for 8 more weeks. Cardiac morphology and function were quantified before/after treatment by echocardiography. After completion of the studies, pigs were euthanized and hearts excised for ex vivo studies. Results: Using IVIS technology, the BDS showed significant retention of SynB1-ELP-p50i in the heart. In the efficacy study, a single IC administration of IC SynB1-ELP-p50i reduced cardiac NF-kB expression, improved cardiac relaxation, and remodeling compared to untreated CKD (Figure). Conclusions: We show for the first time the feasibility of a targeted inhibition of cardiac NF-kB using ELP technology in a translational model of CKD and diastolic dysfunction. Our study may set the stage for a new therapeutic strategy using unique drug-delivery technology never tested before to protect the heart.

Introduction: Type 2 long QT syndrome (LQT2) is caused by loss-of-function variants in the KCNH2 -encoded K v 11.1 potassium channel which is essential for the rapid delayed rectifier channel (I Kr ) that governs phase 3 of cardiac action potential (AP). No current therapies target the molecular cause of LQT2. Methods: A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ~80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA modified by the introduction of synonymous single nucleotide substitutions within the shRNA target site (replacement). The ability of KCNH2-SupRep gene therapy to suppress and replace LQT2-causative variants in KCNH2 was evaluated via heterologous expression in TSA201 cells. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two patients with LQT2 (KCNH2-G604S and -N633S). CRISPR-Cas9 corrected isogenic control iPSC-CMs were made. Both lines were treated with KCNH2-SupRep or non-targeting control shRNA (shCT). FluoVolt voltage dye was used to measure the APD. Results: KCNH2-SupRep achieved mutation-independent suppression of wild-type KCNH2 and both LQT2-causative variants with simultaneous replacement of KCNH2-shIMM as measured by allele-specific qRT-PCR. Treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD at 90% repolarization (APD 90 ) to near isogenic control APD 90 levels (G604S: isogenic controls, 424 + 39 ms and N633S: isogenic controls, 433 + 16 ms) compared to treatment with non-targeting shCT (G604S: SupRep treated, 399±105 ms vs. shCT treated, 577±39 ms, p<0.0001; N633S: SupRep treated, 432±85 ms vs. shCT treated, 483±65 ms, p<0.005). Conclusions: We provide the first proof-of-principle gene therapy for correction of LQT2. Like our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep successfully suppressed and replaced KCNH2 to normal wild-type levels. In TSA201 cells, co-transfection of LQT2 variants and KCNH2-SupRep caused mutation-independent suppression-and-replacement of KCNH2 . In LQT2 iPSC-CMs, KCNH2-SupRep shortened the APD, thereby eliminating the pathognomonic feature of LQT2.

Introduction: LMNA encodes the nuclear envelope proteins lamin A/C that are involved in ageing processes. Mutations in LMNA cause a spectrum of phenotypes which include muscular dystrophy, arrhythmogenic dilated cardiomyopathy, peripheral neuropathy, progeroid syndrome and overlap syndrome. Early diagnosis is crucial to prevent sudden cardiac death. We hypothesized that LMNA mutations result in an ECG biological age older than chronological age. Methods: In this study, we used AI-enabled ECG network to investigate standard digital 12-lead ECGs in genetically characterized LMNA patients. Results: We identified 33 LMNA patients with 331, 12-lead ECG available for analysis. Median age at symptoms onset was 23.5 (range: <1 to 61 years;15 females). Mean follow up time was 86 months (range: <1 to 367 months). Symptoms at onset included muscle weakness (n=10) or cardiac symptoms (n=18) and asymptomatic (n=5). We included a total of 1532 age sex matched controls. We found significant differences in positive age-gap (biologically older than chronological age) comparing LMNA patients (median of 17.5) versus controls (4.9 years; p<0.001, Figure 1 ). AI-ECG age with 10 years or higher age-gap was observed in 73% of LMNA patients compared to 27% in the controls (p<0.001). Consecutive serial analysis for all ECG’s in the study versus predicted age showed faster aging when comparing the rates of age-gap overtime in the LMNA group compared to controls (p<0.0001, Figure 2 ). Moreover, the subset analysis of 23 ECG in the 5 asymptomatic patients versus controls also showed significantly higher AI-ECG age-gap (p=0.004) Conclusions: An AI-ECG algorithm to detect biological age effectively demonstrates accelerated aging in patients with LMNA mutations related to physiological aging. Our findings further validate the role of AI-ECG to detect accelerated aging, expanding previous studies demonstrating higher mortality among individuals with in increased positive age-gap.

Introduction: The process of engaging stem cells towards a cardiopoietic (CP) state was established using a protein-based cocktail consisting of multiple growth factors. Although this regenerative enhancement protocol has been successfully tested in advanced clinical trials, it remains resource-intensive. Recent efforts have focused on streamlining cardiopoiesis through a protein-independent alternative that leverages a transfection-based mRNA approach. Microencapsulated modified messenger RNA (M3RNA) was utilized to deliver brachyury (T), an early mesodermal transcription factor to provoke early lineage specification in pluripotent cells. Hypothesis: Transfection of a human pluripotent stem cell source with T induces early expression of CP transcription factors to accelerate cardiac differentiation. Methods: Human pluripotent stem cells were transfected with T and maintained for 14 days. Expression of the cardiopoietic markers MEF2C, GATA4, and Nkx2.5 was analyzed using RT-qPCR, in tandem with MYH6 at days 10 and 20. Differentiation proficiency was independently corroborated by assessing Mef2c and Nkx2.5 expression by IF. Results: Following transfection with T, Nkx2.5, MEF2C and GATA4 were significantly induced at 72h (60-, 20- and 40-fold, respectively, p<0.01 for all 3 markers vs baseline), reflecting timing of differentiation initiation. At 5 days post differentiation, expression of the cardiac-specific Nkx2.5 increased by 5 fold vs control and the cell cycle regulator CHIR (p<0.01), suggesting successful persistent induction of cardiopoeisis. At 10 and 20 days of differentiation, RNA expression of MYH6 was 10-15 times induced in human pluripotent cells transduced with T (p < 0.0001, and p < 0.001 vs control, respectively), compared to 2 times (p<0.05, p<0.01 vs control, respectively) in CHIR treated cohorts. Accordingly, at 42 days post-induction of differentiation, a greater number of cells expressed MLC2V and cTnT (p<1e-05, <1e-07, respectively vs control), driving a robust beating pattern. Conclusion: This study implements a single gene T M3RNA-based approach to readily induce cardiac differentiation from pluripotent sources, exceeding available commercial platforms.

Background: The primary role of natriuretic peptide receptor 3 (NPR3) is clearance of natriuretic peptides. Genetic variation in NPR3 has been associated with blood pressure regulation and obesity. Despite its importance, variation in NPR3 characterizing coding single nucleotide polymorphisms (SNPs) has not been addressed by resequencing the gene. Objective: We set out to identify and functionally characterize common variation in NPR3 in 3 ethnic groups. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response. Also, rare variants identified by sequencing are enriched in complex disease traits. Methods and Results: DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3. There were 105 polymorphisms identified of which 50 were novel, including 8 nonsynonymous (ns) SNPs of which 7 were novel. Expression constructs for the nsSNPs were created and HEK-293 cells were transfected with constructs for wild type (WT) and the variants. Recombinant proteins were analyzed by quantitative Western blot analysis. A significant reduction in NPR3 immunoreactive protein (60% or less of WT) was observed for the following variants Cys3, Arg146, Val499, and Ser477+Asp521. The Arg146 novel polymorphism resulted in the least protein expression (20% of WT). After treatment of WT and Arg146 cells with proteasome (MG132) and autophagy (3MA) inhibtors, it was determined that autophagy and not proteasomes plays an important role in the degradation of the Arg 146 variant protein. Modeling based on the NPR3 x-ray crystallography structure confirmed that the Arg146 variant were not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: Multiple novel polymorphisms of NPR3 were identified in 3 ethnic groups. There were 4 variant receptors that displayed a significant decrease in NPR3 protein quantity, Arg146 being the variant with the least expression. Degradation of the variant Arg146 was mediated predominantly by autophagy. This SNP could have a significant effect on the metabolism of natriuretic peptides with clinical implications.

Background and Aim: Cardioembolic stroke from non-valvular atrial fibrillation (NVAF) begins with the development of left atrial appendage thrombus (LAAT). Although nearly 15% of patients with NVAF have LAAT by transesophageal echocardiography, the annual stroke rate is 5% on average. The overall aim of this project is to identify variables associated with embolic propensity of LAAT. Materials and Methods: LAAT specimens were obtained during cardiac surgery (MAZE procedure; n=26). Embolized thrombi (n=51) were obtained during surgical embolectomy from lower (n=36) and upper extremities (n=11) and mesenteric arteries (n=4). Platelet-rich regions within sectioned thrombi were sampled by punch biopsy from which total RNA was extracted and analyzed for expression of 5 platelet specific genes: A1 domain of factor XIII, (F13A1); integrin α 2b β 3 (ITGA2B); glycoprotein IX (GP9), platelet factor 4 (PF4), glycoprotein Ib (GP1BB). Gene expression was quantified using TaqMan MGB-probe based quantitative real-time polymerase chain reaction normalized to β 2 macroglobulin using cDNAs reverse transcribed from total RNA. Results: Factor XIII gene expression (Figure ) was significantly lower in embolic (mean fold change 0.08) compared to left atrial thrombi in situ (mean fold change 0.28; p=0.03). Expression of other genes did not show significant change by embolic status. Conclusions: Variable Factor XIII gene expression in thrombi generated during NVAF may in part explain the propensity to embolization. Whereas Factor XIII stabilizes thrombi by crosslinking fibrin without influencing atrial attachment, this data suggests that embolization occurs through fragmentation.

Abstract Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.