Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy

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Abstract

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, single adeno-associated virus (AAV)-packaged mxABE reached A-to-G editing rates up to 84% in vivo, which is at least 20-fold greater compared to rates reported in previous studies using other RNA-editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of wild-type (WT) levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.

Authors

Guoling Li, Ming Jin, Zhifang Li, Qingquan Xiao, Jiajia Lin, Dong Yang, Yuanhua Liu, Xing Wang, Long Xie, Wenqin Ying, Haoqiang Wang, Erwei Zuo, Linyu Shi, Ning Wang, Wanjin Chen, Chunlong Xu, Hui Yang

Glucose- and glutamine-dependent bioenergetics sensitize bone mechanoresponse after unloading by modulating osteocyte calcium dynamics

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Abstract

Disuse osteoporosis is a metabolic bone disease resulted from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during re-ambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca2+ oscillatory dynamics. The unloading-induced compromised osteocytic Ca2+ response to reloading resulted from the HIF-1α/PDK1 axis-mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blocking PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca2+ oscillations in osteocytes. Thus, we provide a mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.

Authors

Xiyu Liu, Zedong Yan, Jing Cai, Dan Wang, Yongqing Yang, Yuanjun Ding, Xi Shao, Xiaoxia Hao, Erping Luo, X. Edward Guo, Peng Luo, Liangliang Shen, Da Jing

LepRb cell-specific deletion of Slug mitigates obesity and NAFLD

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Abstract

Leptin exerts its biological actions by activating LepRb. LepRb signaling impairment and leptin resistance are believed to cause obesity. Transcription factor Slug (also known as Snai2) recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a pro-obesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb cell-specific Slug knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis, accompanied by decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a significantly higher level in SlugΔLepRb than in Slugf/f mice even before their body weight divergence. Conversely, hypothalamic LepRb neuron-specific overexpression of Slug, mediated by AAV-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased LepRb promoter histone 3 lysine-27 methylations, repressive epigenetic marks, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel a previously-unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Authors

Min-Hyun Kim, Yuan Li, Qiantao Zheng, Lin Jiang, Martin G. Myers, Wen-Shu Wu, Liangyou Rui

Long non-coding RNA LEENE promotes angiogenesis and ischemic recovery in diabetes models

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Abstract

Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long non-coding RNAs are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the LncRNA that Enhances Endothelial Nitric oxide synthase Expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression is decreased in diabetic conditions in cultured endothelial cells (EC), mouse hindlimb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduces their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in leene demonstrate impaired angiogenesis and perfusion following hindlimb ischemia. Importantly, overexpression of human LEENE rescues the impaired ischemic response in leene knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promotes transcription of pro-angiogenic genes in ECs, such as KDR and eNOS, potentially by interacting with LEO1, a key component of RNA Polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.

Authors

Xiaofang Tang, Yingjun Luo, Dongqiang Yuan, Riccardo Calandrelli, Naseeb Kaur Malhi, Kiran Sriram, Yifei Miao, Chih Hong Lou, Walter Tsark, Alonso Tapia, Aleysha T. Chen, Guangyu Zhang, Daniel Roeth, Markus Kalkum, Zhao V. Wang, Shu Chien, Rama Natarajan, John P. Cooke, Sheng Zhong, Zhen Bouman Chen

TIGIT inhibition and lenalidomide synergistically promote anti-myeloma immune responses after stem cell transplantation in mice

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Abstract

Autologous stem cell transplantation (ASCT), with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears a potent inhibitor of myeloma-specific immunity and represents a promising checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8 T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical TIGIT Abs that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8 T cell-dependent, anti-myeloma efficacy. Analysis of bone marrow (BM) CD8 T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after stem cell transplantation.

Authors

Simone A. Minnie, Olivia G. Waltner, Kathleen S. Ensbey, Stuart D. Olver, Alika D. Collinge, David P. Sester, Christine R. Schmidt, Samuel R.W. Legg, Shuichiro Takahashi, Nicole S. Nemychenkov, Tomoko Sekiguchi, Gregory Driessens, Ping Zhang, Motoko Koyama, Andrew Spencer, Leona A. Holmberg, Scott N. Furlan, Antiopi Varelias, Geoffrey R. Hill

Mitochondrial dysfunction in macrophages promotes inflammation and suppresses repair after myocardial infarction

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Abstract

Innate immune cells play important roles in tissue injury and repair following acute myocardial infarction (MI). Although reprogramming of macrophage metabolism has been observed during inflammation and resolution phases, the mechanistic link to macrophage phenotype is not fully understood. In this study, we found myeloid specific deletion of mitochondrial Complex I protein Ndufs4 (mKO) reproduced the proinflammatory metabolic profile in macrophages and exaggerated the response to lipopolysacharride. Moreover, mKO mice showed increased mortality, poor scar formation and worsened cardiac function 30 days post-MI. We observed a greater inflammatory response in mKO on day 1 followed by increased cell death of infiltrating macrophages and blunted transition to reparative phase during day 3-7 post-MI. Efferocytosis is markedly impaired in mKO macrophages leading to lower expression of anti-inflammatory cytokine and tissue repair factors, which suppressed the proliferation/activation of myofibroblasts in the infarct area. Mitochondria-targeted ROS scavenging rescued these impairments and improved myofibroblast function in vivo and reduced post-MI mortality in mKO mice. Together these results reveal a novel role of mitochondria in inflammation resolution and tissue repair via modulating efferocytosis and crosstalk with fibroblasts. The findings are significant for post-MI recovery as well as for other inflammatory conditions.

Authors

Shanshan Cai, Mingyue Zhao, Bo Zhou, Akira Yoshii, Darrian Bugg, Outi Villet, Anita Sahu, Gregory S. Olson, Jennifer Davis, Rong Tian

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

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Abstract

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. Trx1 was S-nitrosylated at Cys73 when Cys32-35, the oxidoreductase catalytic center, was oxidized and forms a disulfide bond during ischemia. Unexpectedly, Atg7 Cys545-548 reduced the disulfide bond in Trx1 at Cys32-35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S knock-in mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Authors

Narayani Nagarajan, Shin-ichi Oka, Jihoon Nah, Changgong Wu, Peiyong Zhai, Risa Mukai, Xiaoyong Xu, Sanchita Kashyap, Chun-Yang Huang, Eun-Ah Sung, Wataru Mizushima, Allen Sam Titus, Koichiro Takayama, Youssef Mourad, Jamie Francisco, Tong Liu, Tong Chen, Hong Li, Junichi Sadoshima

A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents

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Abstract

Pain signals are relayed to the brain via a nociceptive system, and in rare situations, this nociceptive system contains genetic variants that can limit pain response. Here we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and further if we can target this region by a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N knock-in mouse using CRISPR/Cas9, we discovered the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral response to chemical noxious stimuli and less hypersensitivity to nerve injury-induced pain, while leaving the response to noxious heat intact. Furthermore, blocking this K710 region in wild-type rodents by a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and rescued pain hypersensitivity induced by nerve injury back to baseline. These findings identify K710 TRPV1 as a discrete site crucial for the control of nociception and provides new insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

Authors

Shufang He, Vanessa O. Zambelli, Pritam Sinharoy, Laura Brabenec, Yang Bian, Freeborn Rwere, Rafaela C.R. Hell, Beatriz Stein Neto, Barbara Hung, Xuan Yu, Meng Zhao, Zhaofei Luo, Chao Wu, Lijun Xu, Katrin J. Svensson, Stacy L. McAllister, Creed M. Stary, Nana-Maria Wagner, Ye Zhang, Eric R. Gross

A non-neutralizing glycoprotein B monoclonal antibody protects against herpes simplex virus disease in mice

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Abstract

There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fc-gamma receptors (FcɣRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2 vaccinated mice and evaluated these mAbs for binding, neutralizing and FcɣR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcɣRIV, a biomarker of ADCC. The cryo-EM structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcɣRIII when engineered as a human IgG1. These results highlight the importance of FcɣR-activating antibodies in protecting against HSV.

Authors

Masayuki Kuraoka, Clare Burn Aschner, Ian W. Windsor, Aakash Mahant Mahant, Scott J. Garforth, Susan Luozheng Kong, Jacqueline M. Achkar, Steven C. Almo, Garnett Kelsoe, Betsy C. Herold

Impact of pre-existing chronic viral infection and reactivation on the development of long COVID

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Abstract

BACKGROUND. The presence and reactivation of chronic viral infections such as Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) have been proposed as potential contributors to Long COVID (LC), but studies in well-characterized post-acute cohorts of individuals with COVID-19 over a longer time course consistent with current case definitions of LC are limited. METHODS. In a cohort of 280 adults with prior SARS-CoV-2 infection, we assessed the presence and types of LC symptoms and prior medical history (including COVID-19 history and HIV status), and performed serological testing for EBV and CMV using a commercial laboratory. We used covariate-adjusted binary logistic regression models to identify independent associations between variables and LC symptoms. RESULTS. We observed that LC symptoms such as fatigue and neurocognitive dysfunction at a median of 4months following initial diagnosis were independently associated with serological evidence suggesting recent EBV reactivation (early antigen-D [EA-D] IgG positivity) or high nuclear antigen (EBNA) IgG levels, but not with ongoing EBV viremia. Serological evidence suggesting recent EBV reactivation (EA-D IgG) was most strongly associated with fatigue (OR 2.12). Underlying HIV infection was also independently associated with neurocognitive LC (OR 2.5). Interestingly, participants who had serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR 0.52). CONCLUSION. Overall, these findings suggest differential effects of chronic viral co-infections on the likelihood of developing LC and predicted distinct syndromic patterns. Further assessment during the acute phase of COVID-19 is warranted. TRIAL REGISTRATION. Long-term Impact of Infection with Novel Coronavirus (LIINC); NCT04362150 FUNDING. This work was supported by the National Institute of Allergy and Infectious Diseases NIH/NIAID 3R01AI141003-03S1 to TJ Henrich, R01AI158013 to M Gandhi and M Spinelli, K24AI145806 to P Hunt, and by the Zuckerberg San Francisco Hospital Department of Medicine and Division of HIV, Infectious Diseases, and Global Medicine. MJP is supported on K23 A137522 and received support from the UCSFBay Area Center for AIDS Research (P30-AI027763).

Authors

Michael J. Peluso, Tyler-Marie Deveau, Sadie E. Munter, Dylan M. Ryder, Amanda M. Buck, Gabriele Beck-Engeser, Fay Chan, Scott Lu, Sarah A. Goldberg, Rebecca Hoh, Viva Tai, Leonel Torres, Nikita S. Iyer, Monika Deswal, Lynn H. Ngo, Melissa Buitrago, Antonio E. Rodriguez, Jessica Y. Chen, Brandon C. Yee, Ahmed Chenna, John W. Winslow, Christos J. Petropoulos, Amelia N. Deitchman, Joanna Hellmuth, Matthew A. Spinelli, Matthew S. Durstenfeld, Priscilla Y. Hsue, John Daniel Kelly, Jeffrey N. Martin, Steven G. Deeks, Peter W. Hunt, Timothy J. Henrich