Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8+ T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.

Precise manipulation of gene expression is pivotal for gene function studies and the optimization of gene therapy. RNA-based gene switches are attractive tools due to their robust tunability by FDA-approved small molecules, the absence of exogenous immunogenic proteins, and the small size for gene delivery vectors such as adeno-associated virus (AAV). However, existing RNA switches only target a single step of gene expression such as transcription or RNA splicing, exhibiting intrinsic limitations in gene regulation. To overcome this issue, this study integrated the aptamer-based polyA regulator (pA), the drug-elicitable alternative splicing module (DreAM) and an engineered translation modulator with conditional upstream open reading frames (uORFs) to construct the DreAM-plus RNA switch. The pA-DreAM concatenation led to 1.5~5.0-fold and 1.2~4.4-fold increase of inducible fold changes than pA and DreAM, respectively. The uORF module further enhanced the switching performance by 1.4~6.3-fold. DreAM-plus-mediated transient transgene expression demonstrated a temporal resolution of about 24 hours and high tissue specificity to liver or heart. Critically, DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, IsDra2 TnpB etc.) that significantly mitigated off-target effects by 1.4~2.8 folds in plasmids, lentivirus and AAV. In a new mouse model with lipid-nanoparticle-delivered pre-existing immunity, DreAM-plus attenuated AAV-delivered Cas-specific CD8 T cell immune toxicity in the liver and the heart. Therefore, multiple RNA switches could be synergistically integrated to build more sophisticated genetic cassettes for enhanced manipulation of gene expression. ### Competing Interest Statement The authors have declared no competing interest. Beijing Natural Science Foundation, F252059 National Natural Science Foundation of China, 82570307 the Science and Technology Plan Project of Tongzhou District, Beijing, WS2025014 Vituner Therapeutics

BACKGROUND:Heart failure with reduced ejection fraction (HFrEF) is characterized by impaired contractility and high mortality. Dysregulation of intracellular ion (ie, Na+/H+ and Ca2+) cycling underlies reduced cardiac contractility. The mechanisms linking myocardial stress to this ion dysregulation remain incompletely understood. Although the metabolic transcription factor SREBP1 (sterol regulatory element-binding protein 1) remodels cardiac metabolism, its role in HFrEF without metabolic comorbidities, particularly regarding ion handling, remains undefined. METHODS:Cardiac tissues from HFrEF patients and mice subjected to transverse aortic constriction (TAC) were analyzed for SREBP1 transactivation of sodium-hydrogen exchanger 3 (NHE3). Cardiomyocyte-specific SREBP1 transgenic (Srebp1a-Tg) and knockdown (Cre-Srebf1f/f) mice were generated. AAV9 vectors carrying Slc9a3 (encoding NHE3), Srebp1a or shRNA against Slc9a3, driven by the cardiomyocyte-specific cTnT promoter, were used to validate the role of the SREBP1-NHE3 in HFrEF. RESULTS:SREBP1 was activated in human hearts with HFrEF because of dilated cardiomyopathy, but without diabetes or hyperlipidemia, and in TAC-induced HFrEF mouse hearts. Srebp1a-Tg mice exhibited impaired cardiac contractility with dysregulated calcium handling in cardiomyocytes without apparent lipid accumulation. Transcriptomics analysis identified increased NHE3 expression in Srebp1a-Tg mice, confirmed by NHE3 upregulation in TAC hearts and human failing hearts. ChIP-seq, ChIP, and promoter reporter assay demonstrated direct transcriptional regulation of SLC9A3 (encoding NHE3) by SREBP1. NHE3 activity was enhanced in cardiomyocytes isolated from Srebp1a-Tg mice or those underwent TAC, whereas cardiomyocyte-specific Srebf1 knockdown in TAC mice reduced NHE3 activity. Cardiomyocyte-specific knockdown of Srebf1 or Slc9a3 restored calcium handling and improved cardiac function in TAC mice. In Srebp1a-Tg mice, NHE3 knockdown alleviated Na+ and Ca2+ overload and rescued cardiac systolic dysfunction. Conversely, NHE3 overexpression caused contractile impairment in both Cre-Srebf1f/f mice and controls, which offset the protective effect because of SREBP1 loss in the context of Na+ and Ca2+ overload. CONCLUSIONS:SREBP1 directly transactivates cardiac NHE3 during the progression of HFrEF, leading to dysregulated calcium handling and impaired contractility, revealing a novel, noncanonical role for SREBP1 in the pathophysiology of heart failure and offering a potential new therapeutic target.

Background Cardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. Method Proximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. Result In vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker γ-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. Conclusion Lmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 82570307 State Key Laboratory of Complex Severe and Rare Diseases, 2025-O-PY-002 Beijing Natural Science Foundation, F252059

BACKGROUND:Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterized by early-onset joint contractures, progressive muscle atrophy, and cardiac abnormalities. Patients with EDMD carrying LMNA sequence variations often exhibit severe cardiac manifestations, including frequent atrioventricular block and ventricular tachycardia. Approximately 20% of those patients may ultimately require heart transplantation. The molecular mechanisms by which LMNA sequence variations lead to EDMD remain unknown. METHODS:Five clinically diagnosed patients with EDMD carrying LMNA sequence variations were recruited. Patient-specific induced pluripotent stem cells (iPSCs) were generated using a nonintegrating Sendai virus. Previously generated iPSCs, derived from 2 healthy donors, were used as controls. The LMNA L204P sequence variation was corrected by genome editing in EDMD iPSC lines to generate isogenic controls. All iPSC-derived cardiomyocytes (iPSC-CMs) were generated using a monolayer-based differentiation protocol. Three-dimensional, strip-format, and force-generating human engineered heart tissues were generated from iPSC-CMs. A knock-in mouse model carrying the Lmna L204P sequence variation was also generated. RESULTS:EDMD-specific iPSC-CMs exhibited a variety of deleterious phenotypes, including disorganized sarcomeres, abnormal nuclear envelope structure, arrhythmias, and contractile dysfunction, when compared with control and gene-corrected iPSC-CMs. Multi-omics analysis further revealed that LMNA directly binds the WNT5A promoter and the Leu204Pro sequence variation reduces chromatin accessibility and WNT5A transcription in EDMD iPSC-CMs. WNT5a (Wnt family member 5a)/RhoA (Ras homolog family member A) signaling inactivation was shown to lead to actin depolymerization and inhibition of actin polymerization in EDMD iPSC-CMs. This results in a deformed nuclear envelope, contractile dysfunction, and impaired trafficking of Cx43 (connexin 43). The impairment of Cx43 trafficking causes reduced distribution of Cx43 at cell-cell borders, contributing to the arrhythmic phenotype in EDMD iPSC-CMs. Pharmacological interventions of exogenous WNT5a supplementation, RhoA activator, or an actin polymerization stabilizer effectively rescued the pathogenic phenotypes of EDMD iPSC-CMs. EDMD engineered heart tissues displayed dysfunctional contractile force generation, which was significantly alleviated by RhoA activator. Lmna L204P heterozygous knock-in mice exhibited impaired cardiac function and developed cardiac arrhythmias in response to sympathetic stress. CONCLUSIONS:We present WNT5a-mediated aberrant actin filament dynamics as a novel mechanism underlying cardiac pathogenic phenotypes in LMNA-related EDMD. Our findings indicate that activating WNT5a/RhoA and stabilizing actin assembly may serve as novel therapeutic strategies for this condition.

Background The genetic variants of LMNA cause an array of diseases that often affect the heart. LMNA-related cardiomyopathy exhibits high-penetrance and early-onset phenotypes that lead to late-stage heart failure or lethal arrhythmia. As a subtype of dilated cardiomyopathy and arrhythmogenic cardiomyopathy, LMNA-related cardiac dysfunction is resistant to existing cardiac therapeutic strategies, leaving a major unmet clinical need in cardiomyopathy management. Aim of Review Here we comprehensively summarize current knowledge about the genetic basis, disease models and pathological mechanisms of LMNA-related cardiomyopathy. Recent translational studies were highlighted to indicate new therapeutic modalities such as gene supplementation, gene silencing and genome editing therapy, which offer potential opportunities to overcome the difficulties in the development of specific drugs for this disease. Key Scientific Concepts of Review LMNA-related cardiomyopathy involves many diverse disease mechanisms that preclude small-molecule drugs that target only a small fraction of the mechanisms. Agreeing to this notion, the first-in-human clinical trial for this disease recently reported futility. By contrast, gene therapy offers the new hope to directly intervene LMNA variants and demonstrates a tremendous potential for breakthrough therapy for this disease. Concepts in this review are also applicable to studies of other genetic diseases that lack effective therapeutics.

Following the publication of this paper, a concerned reader drew to the Editor's attention that a pair of the fluorescence microscopic images shown in Fig. 2A on p. 1518 were strikingly similar to data which had already been accepted for publication in the journal The Anatolian Journal of Cardiology written by different authors, although the same department and research institute were held in common. Upon performing an independent analysis of the data in this paper in the Editorial Office, it also came to light that flow cytometric data in Fig. 2B had already been submitted for publication in another paper to the journal Drug Design, Development and Therapy that featured some of the same authors, although the experimental conditions in the two papers were reported to be different. Owing to the fact that the contentious flow cytometric and fluorescence microscopic data in the above article had apparently already been submitted for publication elsewhere, the Editor of International Journal of Molecular Medicine has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine  45: 1514‑1524, 2020; DOI: 10.3892/ijmm.2020.4513].

Diabetic cardiomyopathy (DbCM) is increasingly prevalent, but intervention targets remain unclear due to the lack of appropriate models and the complexity of risk factors. Here, this work establishes an in vitro assessment system for DbCM function using cardiomyocytes derived from human pluripotent stem cells and engineered heart tissue. This work finds high-fat status in complex diabetes risk factors majorly contributes most to cardiomyocyte death and contractile dysfunction. Notably, PA induced early electrophysiological abnormalities, and lately is associated with cardiac fibrosis, mitochondrial fission, and systolic and diastolic dysfunction at tissue level. Using this in vitro assessment system, this work finds that empagliflozin (EMPA), a first-line glucose-lowering drug, effectively alleviated early PA-induced cardiomyocyte injury. Treatment with EMPA enhanced abnormal diastolic and electrophysiological functions in the PA-hEHT model and significantly reduced endoplasmic reticulum stress, and apoptosis. Furthermore, these promising results are confirmed in a type 2 diabetes mellitus mouse model, reinforcing the potential of EMPA as a therapeutic option to alleviate cardiomyocyte injury under diabetic conditions. These findings suggest that this work has developed an engineered model of diabetic cardiomyopathy that mimics the various stages of lipotoxic myocardial injury and support the use of EMPA as a potential therapeutic option for diabetic or lipotoxic cardiomyopathy.

The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver. Key features • This protocol describes a detailed procedure to construct and validate AAV-based cardiac-specific gene editing in mice. • MicroRNA-122 target sequences (miR122TS) in combination with a Tnnt2 promoter are used to enhance the cardiac specificity in genome editing. • Amplicon sequencing analysis is applied to precisely and sensitively quantify the genome editing efficiency and tissue specificity in mice.

Adeno-associated virus (AAV) is limited by its packaging capacity and the unwanted promoter activity of inverted terminal repeats (ITRs). Here we utilized mini-enhancers to regulate ITR promoters and drive AAV cargo expression in the absence of the canonical promoters, which not only solved the ITR issue but also released more payload for the cargo. As an example, this new design successfully enabled robust, tissue-specific SpCas9 gene editing via a single AAV, which otherwise requires a dual-AAV system due to the large size of SpCas9. ### Competing Interest Statement The authors have declared no competing interest. Noncommunicable Chronic Diseases—National Science and Technology Major Project, 2025ZD0547100, 2024ZD0526500 National Natural Science Foundation of China, 82200265, 82470251, 82470247, 32400942 Beijing Natural Science Foundation, F252059, 25FS1588

Adeno-associated viruses (AAVs) are commonly used for gene therapy, but a clinically relevant method to fine-tune AAV expression is lacking, restricting their therapeutic efficacy and safety. Here we develop the drug-elicitable alternative splicing module (DreAM), which is responsive to risdiplam, a Food and Drug Administration-approved alternative splicing modulator. Risdiplam activated DreAM-regulated AAV expression in a dose-dependent manner with a 2,000-fold inducible change, depending on the dose of risdiplam and the organ of interest. With a temporal resolution of 2 days, DreAM could transiently, reversibly and repeatedly activate AAV expression according to the frequency and duration of risdiplam administration. In this proof-of-concept study, we incorporated DreAM into the cardiomyocyte-specific, liver-detargeted AAV9-Tnnt2-miR122TS vector to transiently activate the cardiomyocyte regeneration factor YAP5SA. A dedifferentiation–proliferation–redifferentiation cycle was established in adult cardiomyocytes, improving cardiac regeneration after myocardial infarction while limiting animal death, AAV9-Tnnt2 expression in the liver and hepatic tumorigenesis. Therefore, DreAM may enhance the efficacy, safety and scope of gene therapy. Chen, Yang et al. present DreAM, the drug-elicitable alternative splicing module, which is responsive to risdiplam, to inducibly regulate adeno-associated virus vector-mediated transgene expression. Proof-of-concept applications in cardiomyocytes revealed its potential for future clinical use in improving cardiac recovery after myocardial infarction.

Metabolic disorders could cause dysregulated glucose and lipid at the systemic level, but how inter-tissue/organ communications contribute to glucolipotoxicity is difficult to dissect in animal models. To solve this problem, myocardium and nerve tissues were modelled by 3D engineered heart tissues (EHTs) and neural organoids (NOs), which were co-cultured in a generalised medium with normal or elevated glucose/fatty acid contents. Morphology, gene expression, cell death and functional assessments detected no apparent alterations of EHTs and NOs in co-culture under normal conditions. By contrast, NOs significantly ameliorated glucolipotoxicity in EHTs. Transcriptomic and protein secretion assays identified the extracellular matrix protein versican as a key molecule that was transferred from NOs into EHTs in the high-glucose/fatty acid condition. Recombinant versican protein treatment was sufficient to reduce glucolipotoxicity in EHTs. Adeno-associated virus-delivered versican overexpression was sufficient to ameliorate cardiac dysfunction in a murine model of diabetic cardiomyopathy. These data provide the proof-of-concept evidence that inter-tissue/organ communications exist in the co-culture of engineered tissues and organoids, which could be systemically studied to explore potential pathological mechanisms and therapeutic strategies for multi-organ diseases in vitro.

Importance Dystroglycanopathies (DGPs) are a group of muscular dystrophies with abnormal glycosylation of dystroglycan. CRPPA is a gene associated with DGPs. Understanding the genetic basis, genotype-phenotype correlations, and population-specific mutations is crucial for accurate diagnosis and genetic counseling.Objective To investigate CRPPA mutations in Chinese pediatric patients with DGPs, analyze genotype-phenotype correlations, and determine whether specific deletions represent founder mutations in this population.Methods Clinical and genetic data of pediatric patients with CRPPA-related DGPs between June 2006 and December 2023 from Peking University First Hospital were collected and analyzed. Muscle biopsy specimens from four patients were examined using immunohistochemistry, immunofluorescence, and electron microscopy. Haplotype analysis was performed to investigate the potential founder mutation.Results Among the 16 patients studied, phenotypes ranged from severe muscle-eye-brain disease to milder limb-girdle muscular dystrophy. Twenty-one pathogenic variants were identified, including five novel variants. A recurrent exon 6-9 deletion emerged as the second most frequent variant (25.0%, 4/16), with haplotype analysis supporting a founder mutation in Chinese patients. At follow-up, most patients remained non-ambulatory, and one patient died of respiratory failure.Interpretation This study broadens the CRPPA mutational spectrum and identifies a founder mutation of exon 6-9 deletion in Chinese patients. These findings have important implications for population-specific screening, diagnosis, and genetic counseling.

Approaches to enhance adeno-associated virus (AAV)-based cardiac gene transfer are the key to successful cardiac gene therapy, but factors influencing AAV transduction remain poorly investigated. This study showed that myocardial infarction (MI) enhanced cardiac AAV transduction, peaking at the third day post-MI in mice. The excessive AAV enrichment at the border zone is due to local vascular permeabilization and cardiomyocyte metabolic remodeling, which is independent of AAV dosage, serotypes and promoters. This effect was harnessed to boost cardiac base editing and improve the outcome of gene therapy for MI in mice. Thus, heart disease itself is a non-negligible factor that alters AAV-based cardiac gene transfer, which provides a new inroad to develop approaches to enhance cardiac gene therapy.

The truncating mutations of LMNA are the major causes of cardiomyopathy. Here we studied 3 mouse models that carry germline, cardiomyocyte-specific, or genetic mosaic Lmna truncating mutations. Whereas the germline mutant manifested cardiac maturation defects, cardiomyocyte-specific mutation triggered pathological hypertrophy. In genetic mosaic analysis, no morphological defects were observed. Three adeno-associated virus (AAV) vectors were applied to addback lamin-A in a ubiquitous, cardiomyocyte-specific, or cardiomyocyte-excluded manner. Strikingly, only ubiquitous and cardiomyocyte-excluded AAV vectors mitigated the cardiac defects. Therefore, Lmna regulates cardiac morphology and function via a non-cell-autonomous mechanism. Noncardiomyocytes are key targets in AAV lamin-A therapy for Lmna-associated cardiac defects.

Obesity is a global issue that warrants the identification of more effective therapeutic targets and a better understanding of the pivotal molecular pathogenesis. Annexin A1 (ANXA1) is known to inhibit phospholipase A2, exhibiting anti-inflammatory activity. However, the specific effects of ANXA1 in obesity and the underlying mechanisms of action remain unclear. Our study reveals that ANXA1 levels are elevated in the adipose tissue of individuals with obesity. Whole-body or adipocyte-specific ANXA1 deletion aggravates obesity and metabolic disorders. ANXA1 levels are higher in stromal vascular fractions (SVFs) than in mature adipocytes. Further investigation into the role of ANXA1 in SVFs reveals that ANXA1 overexpression induces lower numbers of mature adipocytes, while ANXA1-knockout SVFs exhibit the opposite effect. This suggests that ANXA1 plays an important role in adipogenesis. Mechanistically, ANXA1 competes with MYC binding protein 2 (MYCBP2) for interaction with PDZ and LIM domain 7 (PDLIM7). This exposes the MYCBP2-binding site, allowing it to bind more readily to the SMAD family member 4 (SMAD4) and promoting its ubiquitination and degradation. SMAD4 degradation downregulates peroxisome proliferator-activated receptor gamma (PPARγ) transcription and reduces adipogenesis. Treatment with Ac2-26, an active peptide derived from ANXA1, inhibits both adipogenesis and obesity through the mechanism. In conclusion, the molecular mechanism of ANXA1 inhibiting adipogenesis was first uncovered in our study, which is a potential target for obesity prevention and treatment.