In vivo rescue of genetic dilated cardiomyopathy by systemic delivery of nexilin.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

Intervention with metabolites emulating endogenous cell transitions accelerates muscle regeneration in young and aged mice.

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.

Kidney organoid models reveal cilium-autophagy metabolic axis as a therapeutic target for PKD both in vitro and in vivo.

Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

SIRT2 counteracts primate cardiac aging via deacetylation of STAT3 that silences CDKN2B.

Aging is a major risk factor contributing to pathophysiological changes in the heart, yet its intrinsic mechanisms have been largely unexplored in primates. In this study, we investigated the hypertrophic and senescence phenotypes in the hearts of aged cynomolgus monkeys as well as the transcriptomic and proteomic landscapes of young and aged primate hearts. SIRT2 was identified as a key protein decreased in aged monkey hearts, and engineered SIRT2 deficiency in human pluripotent stem cell-derived cardiomyocytes recapitulated key senescence features of primate heart aging. Further investigations revealed that loss of SIRT2 in human cardiomyocytes led to the hyperacetylation of STAT3, which transcriptionally activated CDKN2B and, in turn, triggered cardiomyocyte degeneration. Intra-myocardial injection of lentiviruses expressing SIRT2 ameliorated age-related cardiac dysfunction in mice. Taken together, our study provides valuable resources for decoding primate cardiac aging and identifies the SIRT2-STAT3-CDKN2B regulatory axis as a potential therapeutic target against human cardiac aging and aging-related cardiovascular diseases.

Genome-wide CRISPR activation screening in senescent cells reveals SOX5 as a driver and therapeutic target of rejuvenation.

Our understanding of the molecular basis for cellular senescence remains incomplete, limiting the development of strategies to ameliorate age-related pathologies by preventing stem cell senescence. Here, we performed a genome-wide CRISPR activation (CRISPRa) screening using a human mesenchymal precursor cell (hMPC) model of the progeroid syndrome. We evaluated targets whose activation antagonizes cellular senescence, among which SOX5 outperformed as a top hit. Through decoding the epigenomic landscapes remodeled by overexpressing SOX5, we uncovered its role in resetting the transcription network for geroprotective genes, including HMGB2. Mechanistically, SOX5 binding elevated the enhancer activity of HMGB2 with increased levels of H3K27ac and H3K4me1, raising HMGB2 expression so as to promote rejuvenation. Furthermore, gene therapy with lentiviruses carrying SOX5 or HMGB2 rejuvenated cartilage and alleviated osteoarthritis in aged mice. Our study generated a comprehensive list of rejuvenators, pinpointing SOX5 as a potent driver for rejuvenation both in vitro and in vivo.

A single-cell transcriptomic atlas of exercise-induced anti-inflammatory and geroprotective effects across the body.

Exercise benefits the whole organism, yet, how tissues across the body orchestrally respond to exercise remains enigmatic. Here, in young and old mice, with or without exercise, and exposed to infectious injury, we characterized the phenotypic and molecular adaptations to a 12-month exercise across 14 tissues/organs at single-cell resolution. Overall, exercise protects tissues from infectious injury, although more effectively in young animals, and benefits aged individuals in terms of inflammaging suppression and tissue rejuvenation, with structural improvement in the central nervous system and systemic vasculature being the most prominent. In vascular endothelial cells, we found that readjusting the rhythmic machinery via the core circadian clock protein BMAL1 delayed senescence and facilitated recovery from infectious damage, recapitulating the beneficial effects of exercise. Our study underscores the effect of exercise in reconstituting the youthful circadian clock network and provides a foundation for further investigating the interplay between exercise, aging, and immune challenges across the whole organism.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Resurrection of endogenous retroviruses during aging reinforces senescence.

Whether and how certain transposable elements with viral origins, such as endogenous retroviruses (ERVs) dormant in our genomes, can become awakened and contribute to the aging process is largely unknown. In human senescent cells, we found that HERVK (HML-2), the most recently integrated human ERVs, are unlocked to transcribe viral genes and produce retrovirus-like particles (RVLPs). These HERVK RVLPs constitute a transmissible message to elicit senescence phenotypes in young cells, which can be blocked by neutralizing antibodies. The activation of ERVs was also observed in organs of aged primates and mice as well as in human tissues and serum from the elderly. Their repression alleviates cellular senescence and tissue degeneration and, to some extent, organismal aging. These findings indicate that the resurrection of ERVs is a hallmark and driving force of cellular senescence and tissue aging.

LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes.

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Myc Supports Self-Renewal of Basal Cells in the Esophageal Epithelium.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice.

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming.

Incidence of COVID-19 in 902 Patients With Immunomediated Inflammatory Diseases Treated With Biologics and Targeted Synthetic Disease-Modifying Antirheumatic Drugs-Findings From the BIOCOVID Study.

OBJECTIVES: The aim of this study was to examine the incidence of coronavirus disease 2019 (COVID-19) among patients with immunomediated inflammatory diseases (IMIDs) treated with biologic or targeted synthetic disease-modifying antirheumatic drugs (bDMARDs and tsDMARDs) and to evaluate the influence of either IMIDs or related therapies on the incidence and evolution of COVID-19. METHODS: This observational, cross-sectional study was conducted from January 31, 2020, to May 15, 2020. Data of 902 patients were obtained from clinical records in hospitals, primary care units, and community pharmacies. Inclusion criteria were adults with IMIDs treated with bDMARDs or tsDMARDs who started therapy 3 months prior to study commencement. Patients with poor adherence to treatments were excluded. COVID-19 was classified as "definitive" (severe acute respiratory syndrome coronavirus 2 polymerase chain reaction [PCR]-positive), "possible" (characteristic symptoms and negative PCR), and "suspected" (characteristic symptoms but PCR not performed). RESULTS: COVID-19 was diagnosed in 70 patients (11 definitive, 19 possible, and 40 suspected). The cumulative incidence of definitive COVID-19 was 1.2%. When considering all cases, the incidence was 7.8%. Patients on biosimilars tumor necrosis factor blockers were more likely to have a diagnosis of COVID-19 (odds ratio, 2.308; p < 0.001). Patients on anti-B-cell therapies had a lower incidence of infections (p = 0.046). Low rates of hospitalization (14.3%), pneumonia (14.3%), death (2.9%), or thrombosis (2.9%) were observed, and 94.3% of patients recovered. CONCLUSIONS: The cumulative incidence of confirmed cases of COVID-19 was similar to the general population, with generally low hospitalization, intensive care management, and mortality rates. COVID-19 incidence was less frequent in patients with more severe immunosuppression.

In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche.

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Simultaneous detection and mutation surveillance of SARS-CoV-2 and multiple respiratory viruses by rapid field-deployable sequencing.

BACKGROUND: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. METHODS: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. FINDINGS: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples. CONCLUSIONS: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. FUNDING: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence.

Understanding the genetic and epigenetic bases of cellular senescence is instrumental in developing interventions to slow aging. We performed genome-wide CRISPR-Cas9-based screens using two types of human mesenchymal precursor cells (hMPCs) exhibiting accelerated senescence. The hMPCs were derived from human embryonic stem cells carrying the pathogenic mutations that cause the accelerated aging diseases Werner syndrome and Hutchinson-Gilford progeria syndrome. Genes whose deficiency alleviated cellular senescence were identified, including KAT7, a histone acetyltransferase, which ranked as a top hit in both progeroid hMPC models. Inactivation of KAT7 decreased histone H3 lysine 14 acetylation, repressed p15INK4b transcription, and alleviated hMPC senescence. Moreover, lentiviral vectors encoding Cas9/sg-Kat7, given intravenously, alleviated hepatocyte senescence and liver aging and extended life span in physiologically aged mice as well as progeroid Zmpste24-/- mice that exhibit a premature aging phenotype. CRISPR-Cas9-based genetic screening is a robust method for systematically uncovering senescence genes such as KAT7, which may represent a therapeutic target for developing aging interventions.