The PD-1-PD-L1 pathway maintains an immunosuppressive environment essential for neonatal heart regeneration.

The adult mouse heart responds to injury by scarring with consequent loss of contractile function, whereas the neonatal heart possesses the ability to regenerate. Activation of the immune system is among the first events upon tissue injury. It has been shown that immune response kinetics differ between regeneration and pathological remodeling, yet the underlying mechanisms of the distinct immune reactions during tissue healing remain unclear. Here we show that the immunomodulatory PD-1-PD-L1 pathway is highly active in regenerative neonatal hearts but rapidly silenced later in life. Deletion of the PD-1 receptor or inactivation of its ligand PD-L1 prevented regeneration of neonatal hearts after injury. Disruption of the pathway during neonatal cardiac injury led to increased inflammation and aberrant T cell activation, which ultimately impaired cardiac regeneration. Our findings reveal an immunomodulatory and cardioprotective role for the PD-1-PD-L1 pathway in heart regeneration and offer potential avenues for the control of adult tissue regeneration.

Coactivator condensation drives cardiovascular cell lineage specification.

During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.

Patterns of neurological adverse events among a retrospective cohort of patients receiving immune checkpoint inhibitors.

Aim: Neurological adverse events (NAEs) are infrequent immune checkpoint inhibitor (ICI) outcomes poorly characterized in extant research, complicating their clinical management. Methods: This study characterized the frequency, severity, patterning and timing of NAEs using a large retrospective registry, including all patients who received at least one dose of an ICI from 2/1/2011-4/7/2022 within our health network. Results: Among 3137 patients, there were 54 NAEs (1.72% any grade; 0.8% grade 3-4). Most NAEs were peripheral (57.4%) versus central (42.6%). Melanoma and renal cell carcinoma were significantly associated with NAEs. Conclusion: The incidence of NAEs was rare though higher than many prior case estimates; the timing was consistent with other AEs. NAEs frequently occurred in tumor types known to favor brain metastases.

Immune checkpoint inhibitors are new drugs for cancer. They boost your body's defenses to fight cancer cells. These drugs can be used alone or with other cancer treatments. Most people are okay with these medicines, but some might have problems in different parts of the body. This can be tricky to figure out. Rarely, there can be issues in the brain or nerves. These side effects are rare, happening in about 2 in every 100 people who use the drugs. They are more common in certain cancers like melanoma and kidney cancer. As doctors learn more about these side effects, they can better predict, treat, and prevent them.

CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

Permanent standard time is the optimal choice for health and safety: an American Academy of Sleep Medicine position statement.

The period of the year from spring to fall, when clocks in most parts of the United States are set one hour ahead of standard time, is called daylight saving time, and its beginning and ending dates and times are set by federal law. The human biological clock is regulated by the timing of light and darkness, which then dictates sleep and wake rhythms. In daily life, the timing of exposure to light is generally linked to the social clock. When the solar clock is misaligned with the social clock, desynchronization occurs between the internal circadian rhythm and the social clock. The yearly change between standard time and daylight saving time introduces this misalignment, which has been associated with risks to physical and mental health and safety, as well as risks to public health. In 2020, the American Academy of Sleep Medicine (AASM) published a position statement advocating for the elimination of seasonal time changes, suggesting that evidence best supports the adoption of year-round standard time. This updated statement cites new evidence and support for permanent standard time. It is the position of the AASM that the United States should eliminate seasonal time changes in favor of permanent standard time, which aligns best with human circadian biology. Evidence supports the distinct benefits of standard time for health and safety, while also underscoring the potential harms that result from seasonal time changes to and from daylight saving time. CITATION: Rishi MA, Cheng JY, Strang AR, et al. Permanent standard time is the optimal choice for health and safety: an American Academy of Sleep Medicine position statement. J Clin Sleep Med. 2024;20(1):121-125.

SARS-CoV-2 Humoral Immune Responses in Convalescent Individuals Over 12 Months Reveal Severity-Dependent Antibody Dynamics.

Background: Understanding the kinetics and longevity of antibody responses to SARS-CoV-2 is critical to informing strategies toward reducing Coronavirus disease 2019 (COVID-19) reinfections, and improving vaccination and therapy approaches. Methods: We evaluated antibody titers against SARS-CoV-2 nucleocapsid (N), spike (S), and receptor binding domain (RBD) of spike in 98 convalescent participants who experienced asymptomatic, mild, moderate or severe COVID-19 disease and in 17 non-vaccinated, non-infected controls, using four different antibody assays. Participants were sampled longitudinally at 1, 3, 6, and 12 months post-SARS-CoV-2 positive PCR test. Findings: Increasing acute COVID-19 disease severity correlated with higher anti-N and anti-RBD antibody titers throughout 12 months post-infection. Anti-N and anti-RBD titers declined over time in all participants, with the exception of increased anti-RBD titers post-vaccination, and the decay rates were faster in hospitalized compared to non-hospitalized participants. <50% of participants retained anti-N titers above control levels at 12 months, with non-hospitalized participants falling below control levels sooner. Nearly all hospitalized and non-hospitalized participants maintained anti-RBD titers above controls for up to 12 months, suggesting longevity of protection against severe reinfections. Nonetheless, by 6 months, few participants retained >50% of their 1-month anti-N or anti-RBD titers. Vaccine-induced increases in anti-RBD titers were greater in non-hospitalized relative to hospitalized participants. Early convalescent antibody titers correlated with age, but no association was observed between Post-Acute Sequelae of SARS-CoV-2 infection (PASC) status or acute steroid treatment and convalescent antibody titers. Interpretation: Hospitalized participants developed higher anti-SARS-CoV-2 antibody titers relative to non-hospitalized participants, a difference that persisted throughout 12 months, despite the faster decline in titers in hospitalized participants. In both groups, while anti-N titers fell below control levels for at least half of the participants, anti-RBD titers remained above control levels for almost all participants over 12 months, demonstrating generation of long-lived antibody responses known to correlate with protection from severe disease across COVID-19 severities. Overall, our findings contribute to the evolving understanding of COVID-19 antibody dynamics. Funding: Austin Public Health, NIAAA, Babson Diagnostics, Dell Medical School Startup.

Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development.

Cardiomyocytes are highly metabolic cells responsible for generating the contractile force in the heart. During fetal development and regeneration, these cells actively divide but lose their proliferative activity in adulthood. The mechanisms that coordinate their metabolism and proliferation are not fully understood. Here, we study the role of the transcription factor NFYa in developing mouse hearts. Loss of NFYa alters cardiomyocyte composition, causing a decrease in immature regenerative cells and an increase in trabecular and mature cardiomyocytes, as identified by spatial and single-cell transcriptome analyses. NFYa-deleted cardiomyocytes exhibited reduced proliferation and impaired mitochondrial metabolism, leading to cardiac growth defects and embryonic death. NFYa, interacting with cofactor SP2, activates genes linking metabolism and proliferation at the transcription level. Our study identifies a nodal role of NFYa in regulating prenatal cardiac growth and a previously unrecognized transcriptional control mechanism of heart metabolism, highlighting the importance of mitochondrial metabolism during heart development and regeneration.

DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters.

BACKGROUND: The p.Arg14del variant of the PLN (phospholamban) gene causes cardiomyopathy, leading to severe heart failure. Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease. Dwarf open reading frame (DWORF) has been shown to counteract PLN regulatory calcium handling function in the sarco/endoplasmic reticulum (S/ER). Here, we investigated the potential disease-modulating action of DWORF in this cardiomyopathy and its effects on calcium handling and PLN aggregation. METHODS: We studied a PLN-R14del mouse model, which develops cardiomyopathy with similar characteristics as human patients, and explored whether cardiac DWORF overexpression could delay cardiac deterioration. To this end, R14Δ/Δ (homozygous PLN-R14del) mice carrying the DWORF transgene (R14Δ/ΔDWORFTg [R14Δ/Δ mice carrying the DWORF transgene]) were used. RESULTS: DWORF expression was suppressed in hearts of R14Δ/Δ mice with severe heart failure. Restoration of DWORF expression in R14Δ/Δ mice delayed cardiac fibrosis and heart failure and increased life span >2-fold (from 8 to 18 weeks). DWORF accelerated sarcoplasmic reticulum calcium reuptake and relaxation in isolated cardiomyocytes with wild-type PLN, but in R14Δ/Δ cardiomyocytes, sarcoplasmic reticulum calcium reuptake and relaxation were already enhanced, and no differences were detected between R14Δ/Δ and R14Δ/ΔDWORFTg. Rather, DWORF overexpression delayed the appearance and formation of large pathogenic perinuclear PLN clusters. Careful examination revealed colocalization of sarcoplasmic reticulum markers with these PLN clusters in both R14Δ/Δ mice and human p.Arg14del PLN heart tissue, and hence these previously termed aggregates are comprised of abnormal organized S/ER. This abnormal S/ER organization in PLN-R14del cardiomyopathy contributes to cardiomyocyte cell loss and replacement fibrosis, consequently resulting in cardiac dysfunction. CONCLUSIONS: Disorganized S/ER is a major characteristic of PLN-R14del cardiomyopathy in humans and mice and results in cardiomyocyte death. DWORF overexpression delayed PLN-R14del cardiomyopathy progression and extended life span in R14Δ/Δ mice, by reducing abnormal S/ER clusters.

Cryo-EM structures of Myomaker reveal a molecular basis for myoblast fusion.

The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane protein, is essential for myoblast fusion. Here we report the cryo-EM structures of mouse Myomaker (mMymk) and Ciona robusta Myomaker (cMymk). Myomaker contains seven transmembrane helices (TMs) that adopt a G-protein-coupled receptor-like fold. TMs 2-4 form a dimeric interface, while TMs 3 and 5-7 create a lipid-binding site that holds the polar head of a phospholipid and allows the alkyl tails to insert into Myomaker. The similarity of cMymk and mMymk suggests a conserved Myomaker-mediated cell fusion mechanism across evolutionarily distant species. Functional analyses demonstrate the essentiality of the dimeric interface and the lipid-binding site for fusogenic activity, and heterologous cell-cell fusion assays show the importance of transcellular interactions of Myomaker protomers for myoblast fusion. Together, our findings provide structural and functional insights into the process of myoblast fusion.

Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.

Calcium Signaling during Cortical Apical Dendrite Initiation: A Role for Cajal-Retzius Neurons.

The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This transformation occurs in the cortical marginal zone (MZ), a layer that contains the Cajal-Retzius neurons and their axonal projections. Cajal-Retzius neurons (CRNs) are well known for their critical role in secreting Reelin, a glycoprotein that controls dendritogenesis and cell positioning in many regions of the developing brain. In this study, we examine the possibility that CRNs in the MZ may provide additional signals to arriving CPNs, that may promote the maturation of CPNs and thus shape the development of the cortex. We use whole embryonic hemisphere explants and multiphoton microscopy to confirm that CRNs display intracellular calcium transients of <1-min duration and high amplitude during early corticogenesis. In contrast, developing CPNs do not show high-amplitude calcium transients, but instead show a steady increase in intracellular calcium that begins at the time of dendritic initiation, when the leading process of the migrating CPN is encountering the MZ. The possible existence of CRN to CPN communication was revealed by the application of veratridine, a sodium channel activator, which has been shown to preferentially stimulate more mature cells in the MZ at an early developmental time. Surprisingly, veratridine application also triggers large calcium transients in CPNs, which can be partially blocked by a cocktail of antagonists that block glutamate and glycine receptor activation. These findings outline a model in which CRN spontaneous activity triggers the release of glutamate and glycine, neurotransmitters that can trigger intracellular calcium elevations in CPNs. These elevations begin as CPNs initiate dendritogenesis and continue as waves in the post-migratory cells. Moreover, we show that the pharmacological blockade of glutamatergic signaling disrupts migration, while forced expression of a bacterial voltage-gated calcium channel (CavMr) in the migrating neurons promotes dendritic growth and migration arrest. The identification of CRN to CPN signaling during early development provides insight into the observation that many autism-linked genes encode synaptic proteins that, paradoxically, are expressed in the developing cortex well before the appearance of synapses and the establishment of functional circuits.

Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy.

Mutations in genes encoding nuclear envelope proteins lead to diseases known as nuclear envelopathies, characterized by skeletal muscle and heart abnormalities, such as Emery-Dreifuss muscular dystrophy (EDMD). The tissue-specific role of the nuclear envelope in the etiology of these diseases has not been extensively explored. We previously showed that global deletion of the muscle-specific nuclear envelope protein NET39 in mice leads to neonatal lethality due to skeletal muscle dysfunction. To study the potential role of the Net39 gene in adulthood, we generated a muscle-specific conditional knockout (cKO) of Net39 in mice. cKO mice recapitulated key skeletal muscle features of EDMD, including muscle wasting, impaired muscle contractility, abnormal myonuclear morphology, and DNA damage. The loss of Net39 rendered myoblasts hypersensitive to mechanical stretch, resulting in stretch-induced DNA damage. Net39 was downregulated in a mouse model of congenital myopathy, and restoration of Net39 expression through AAV gene delivery extended life span and ameliorated muscle abnormalities. These findings establish NET39 as a direct contributor to the pathogenesis of EDMD that acts by protecting against mechanical stress and DNA damage.

Sex-specific associations between daytime sleepiness, chronic diseases and mortality in obstructive sleep apnea.

Objective: Excessive daytime sleepiness (EDS) is common in obstructive sleep apnea (OSA) and has been linked to adverse outcomes, albeit inconsistently. Furthermore, whether the prognostic impact of EDS differs as a function of sex is unclear. We aimed to assess the associations between EDS and chronic diseases and mortality in men and women with OSA. Methods: Newly-diagnosed adult OSA patients who underwent sleep evaluation at Mayo Clinic between November 2009 and April 2017 and completed the Epworth Sleepiness Scale (ESS) for assessment of perceived sleepiness (N = 14,823) were included. Multivariable-adjusted regression models were used to investigate the relationships between sleepiness, with ESS modeled as a binary (ESS > 10) and as a continuous variable, and chronic diseases and all-cause mortality. Results: In cross-sectional analysis, ESS > 10 was independently associated with lower risk of hypertension in male OSA patients (odds ratio [OR], 95% confidence interval [CI]: 0.76, 0.69-0.83) and with higher risk of diabetes mellitus in both OSA men (OR, 1.17, 95% CI 1.05-1.31) and women (OR 1.26, 95% CI 1.10-1.45). Sex-specific curvilinear relations between ESS score and depression and cancer were noted. After a median 6.2 (4.5-8.1) years of follow-up, the hazard ratio for all-cause death in OSA women with ESS > 10 compared to those with ESS ≤ 10 was 1.24 (95% CI 1.05-1.47), after adjusting for demographics, sleep characteristics and comorbidities at baseline. In men, sleepiness was not associated with mortality. Conclusion: The implications of EDS for morbidity and mortality risk in OSA are sex-dependent, with hypersomnolence being independently associated with greater vulnerability to premature death only in female patients. Efforts to mitigate mortality risk and restore daytime vigilance in women with OSA should be prioritized.

Opposing gene regulatory programs governing myofiber development and maturation revealed at single nucleus resolution.

Skeletal muscle fibers express distinct gene programs during development and maturation, but the underlying gene regulatory networks that confer stage-specific myofiber properties remain unknown. To decipher these distinctive gene programs and how they respond to neural activity, we generated a combined multi-omic single-nucleus RNA-seq and ATAC-seq atlas of mouse skeletal muscle development at multiple stages of embryonic, fetal, and postnatal life. We found that Myogenin, Klf5, and Tead4 form a transcriptional complex that synergistically activates the expression of muscle genes in developing myofibers. During myofiber maturation, the transcription factor Maf acts as a transcriptional switch to activate the mature fast muscle gene program. In skeletal muscles of mutant mice lacking voltage-gated L-type Ca2+ channels (Cav1.1), Maf expression and myofiber maturation are impaired. These findings provide a transcriptional atlas of muscle development and reveal genetic links between myofiber formation, maturation, and contraction.

Delayed cortical development in mice with a neural specific deletion of ß1 integrin.

The adhesion systems employed by migrating cortical neurons are not well understood. Genetic deletion studies of focal adhesion kinase (FAK) and paxillin in mice suggested that these classical focal adhesion molecules control the morphology and speed of cortical neuron migration, but whether ß1 integrins also regulate migration morphology and speed is not known. We hypothesized that a ß1 integrin adhesion complex is required for proper neuronal migration and for proper cortical development. To test this, we have specifically deleted ß1 integrin from postmitotic migrating and differentiating neurons by crossing conditional ß1 integrin floxed mice into the NEX-Cre transgenic line. Similar to our prior findings with conditional paxillin deficiency, we found that both homozygous and heterozygous deletion of ß1 integrin causes transient mispositioning of cortical neurons in the developing cortex when analyzed pre- and perinatally. Paxillin and ß1 integrin colocalize in the migrating neurons and deletion of paxillin in the migrating neuron causes an overall reduction of the ß1 integrin immunofluorescence signal and reduction in the number of activated ß1 integrin puncta in the migrating neurons. These findings suggest that these molecules may form a functional complex in migrating neurons. Similarly, there was an overall reduced number of paxillin+ puncta in the ß1 integrin deficient neurons, despite the normal distribution of FAK and Cx26, a connexin required for cortical migration. The double knockout of paxillin and ß1 integrin produces a cortical malpositioning phenotype similar to the paxillin or ß1 integrin single knockouts, as would be expected if paxillin and ß1 integrin function on a common pathway. Importantly, an isolation-induced pup vocalization test showed that ß1 integrin mutants produced a significantly smaller number of calls compared to their littermate controls when analyzed at postnatal day 4 (P4) and revealed a several days trend in reduced vocalization development compared to controls. The current study establishes a role for ß1 integrin in cortical development and suggests that ß1 integrin deficiency leads to migration and neurodevelopmental delays.

Single-swap editing for the correction of common Duchenne muscular dystrophy mutations.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease of progressive muscle weakness and wasting caused by the absence of dystrophin protein. Current gene therapy approaches using antisense oligonucleotides require lifelong dosing and have limited efficacy in restoring dystrophin production. A gene editing approach could permanently correct the genome and restore dystrophin protein expression. Here, we describe single-swap editing, in which an adenine base editor edits a single base pair at a splice donor site or splice acceptor site to enable exon skipping or reframing. In human induced pluripotent stem cell-derived cardiomyocytes, we demonstrate that single-swap editing can enable beneficial exon skipping or reframing for the three most therapeutically relevant exons-DMD exons 45, 51, and 53-which could be beneficial for 30% of all DMD patients. Furthermore, an adeno-associated virus delivery method for base editing components can efficiently restore dystrophin production locally and systemically in skeletal and cardiac muscles of a DMD mouse model containing a deletion of Dmd exon 44. Our studies demonstrate single-swap editing as a potential gene editing therapy for common DMD mutations.

CRISPR-Editing Therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.

TWIST2-mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma in children that resembles developing skeletal muscle. Unlike normal muscle cells, RMS cells fail to differentiate despite expression of the myogenic determination protein MYOD. The TWIST2 transcription factor is frequently overexpressed in fusion-negative RMS (FN-RMS). TWIST2 blocks differentiation by inhibiting MYOD activity in myoblasts, but its role in FN-RMS pathogenesis is incompletely understood. Here, we show that knockdown of TWIST2 enables FN-RMS cells to exit the cell cycle and undergo terminal myogenesis. TWIST2 knockdown also substantially reduces tumor growth in a mouse xenograft model of FN-RMS. Mechanistically, TWIST2 controls H3K27 acetylation at distal enhancers by interacting with the chromatin remodelers SMARCA4 and CHD3 to activate growth-related target genes and repress myogenesis-related target genes. These findings provide insights into the role of TWIST2 in maintaining an undifferentiated and tumorigenic state of FN-RMS and highlight the potential of suppressing TWIST2-regulated pathways to treat FN-RMS.