Meeting Report: 2023 Muscular Dystrophy Association Summit on 'Safety and Challenges in Gene Therapy of Neuromuscular Diseases'.

 This meeting report summarizes the presentations and discussions held at the summit on Challenges in Gene Therapy hosted by the Muscular Dystrophy Association (MDA) in 2023. Topics covered include safety issues, mitigation strategies and practical considerations pertaining to the clinical translation of gene therapies for neuromuscular disease. The listing of actionable recommendations will assist in overall efforts in the field to achieve safe and efficacious translation of gene therapies for neuromuscular disease patients.

Airway basal stem cells are necessary for the maintenance of functional intraepithelial airway macrophages.

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.

BACKGROUND: ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance. METHODS: Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied. RESULTS: Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses. CONCLUSIONS: ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.

Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

Potential limitations of microdystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (microdystrophins) are underway for Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with 4 microdystrophin constructs (3 in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using AAV doses comparable with those in clinical trials. We achieved high levels of microdystrophin expression in striated muscles with cardiac expression approximately 10-fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease occurred with 2 of the microdystrophins. The detrimental cardiac effect appears to be caused by variable competition (dependent on microdystrophin design and expression level) between microdystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-microdystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, these findings suggest that microdystrophin treatments need to avoid excessively high levels of expression in the heart and that cardiac function should be carefully monitored in these patients.

Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers.

The mammalian olfactory neuronal lineage is regenerative, and accordingly, maintains a population of pluripotent cells that replenish olfactory sensory neurons and other olfactory cell types during the life of the animal. Moreover, in response to acute injury, the early transit amplifying cells along the olfactory sensory neuronal lineage are able to de-differentiate to shift resources in support of tissue restoration. In order to further explore plasticity of various cellular stages along the olfactory sensory neuronal lineage, we challenged the epigenetic stability of two olfactory placode-derived cell lines that model immature olfactory sensory neuronal stages. We found that perturbation of the Ehmt2 chromatin modifier transformed the growth properties, morphology, and gene expression profiles towards states with several stem cell characteristics. This transformation was dependent on continued expression of the large T-antigen, and was enhanced by Sox2 over-expression. These findings may provide momentum for exploring inherent cellular plasticity within early cell types of the olfactory lineage, as well as potentially add to our knowledge of cellular reprogramming. SUMMARY STATEMENT: Discovering how epigenetic modifications influence olfactory neuronal lineage plasticity offers insights into regenerative potential and cellular reprogramming.

The proteomic landscape of genotoxic stress-induced micronuclei.

Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.

Investigating adverse genomic and regulatory changes caused by replacement of the full-length CFTR cDNA using Cas9 and AAV.

A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in ∼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.

The characteristics of pre-existing humoral imprint determine efficacy of S. aureus vaccines and support alternative vaccine approaches.

The failure of the Staphylococcus aureus (SA) IsdB vaccine trial can be explained by the recall of non-protective immune imprints from prior SA exposure. Here, we investigate natural human SA humoral imprints to understand their broader impact on SA immunizations. We show that antibody responses against SA cell-wall-associated antigens (CWAs) are non-opsonic, while antibodies against SA toxins are neutralizing. Importantly, the protective characteristics of the antibody imprints accurately predict the failure of corresponding vaccines against CWAs and support vaccination against toxins. In passive immunization platforms, natural anti-SA human antibodies reduce the efficacy of the human monoclonal antibodies suvratoxumab and tefibazumab, consistent with the results of their respective clinical trials. Strikingly, in the absence of specific humoral memory responses, active immunizations are efficacious in both naive and SA-experienced mice. Overall, our study points to a practical and predictive approach to evaluate and develop SA vaccines based on pre-existing humoral imprint characteristics.

Analysis of controllable risk factors of osteoporotic vertebral fractures / 中华健康管理学杂志

Objective To analyze the controlled risk factors of osteoporotic vertebral fractures, and determine the clinical value for the management of risk factors. Methods 626 cases of postmenopausal women were selected, age, height, weight and bone mineral density(BMD) of patients were collected.The Tetrax balance test system was used to assess the fall risks.Vertebral changes of patients were evaluated through X ray of thoracolumbar lateral. The clinical data were analyzed and compared between vertebral fracture group and non-vertebral fracture group. Spearman test was used to analyze the correlation between vertebral fracture and age, height, weight, body mass index(BMI), the risk of fall, body fat and BMD. Results 328 patients presented with osteoporotic vertebral fractures, while the other 298 cases did not suffer from osteoporotic vertebral fractures. There were 426 vertebral bodies involved. Comparing to non-vertebral fracture group, the vertebral fracture group showed higher age [(68.67±9.29)years vs.(63.04± 9.30)years], lower height[(151.10 ± 4.39)cm vs.(154.90 ± 5.86)cm], lower bone mineral density[lumbar spine BMD (0.85 ± 0.16)g/cm2 vs.(0.93 ± 0.17)g/cm2, hip spine BMD (0.72 ± 0.18)g/cm2 vs.(0.81 ± 0.13)g/cm2],higher body fat [(41.30 ± 5.20)％vs.(36.30 ± 5.90)％] and higher fall risk (41.38 ± 25.79 vs. 36.20 ± 26.22) ( P<0.001). While there were no statistical differences in weight and BMI between the two groups, age, height, BMI, body fat, fall risk, lumbar spine and hip BMD were significantly correlated with vertebral fracture (r=0.358,-0.323, 0.169, 0.186, 0.135,-0.398,-0.364, respectively, all P<0.001). Conclusions Decreased bone mineral density, increased age, abdomen fat content and increased fall risk, are the risk factors of osteoporotic vertebral fractures.