Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated α-Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.
Fabian Braun, Ahmed Abed, Dominik Sellung, Manuel Rogg, Mathias Woidy, Oysten Eikrem, Nicola Wanner, Jessica Gambardella, Sandra D. Laufer, Fabian Haas, Milagros N. Wong, Bernhard Dumoulin, Paula Rischke, Anne K. Mühlig, Wiebke Sachs, Katharina von Cossel, Kristina Schulz, Nicole Muschol, Sören W. Gersting, Ania C. Muntau, Oliver Kretz, Oliver Hahn, Markus M. Rinschen, Michael Mauer, Tillmann Bork, Florian Grahammer, Wei Liang, Thorsten Eierhoff, Winfried Römer, Arne Hansen, Catherine Meyer-Schwesinger, Guido Iaccarino, Camilla Tøndel, Hans-Peter Marti, Behzad Najafian, Victor G. Puelles, Christoph Schell, Tobias B. Huber
Programmed death ligand 1 (PD-L1) is an immune checkpoint protein frequently expressed in human cancers, which contributes to immune evasion through its binding to PD-1 on activated T cells. Unveiling the mechanisms underlying PD-L1 expression is essential for understanding the impacts of immunosuppressive microenvironment, and also crucial for the purpose of re-boosting anti-tumour immunity. However, how PD-L1 is regulated, particularly at translational levels, remains largely unknown. Here, we discovered that a lncRNA, HIF-1α inhibitor at translation level (HITT), was transactivated by E2F1 under interferon-γ stimulation. It bound and co-ordinated with Regulator of G Protein Signalling 2 (RGS2) in binding to the 5ʹ-untranslated region (UTR) of PD-L1, resulting in reduced PD-L1 translation. HITT expression enhanced T cell-mediated cytotoxicity both in vitro and in vivo in a PD-L1 dependent manner. The clinical correlation between HITT/PD-L1, RGS2/PD-L1 expression was also detected in breast cancer tissues. Together, these findings demonstrate the role of HITT in antitumour T cell immunity, highlighting activation of HITT as a potential therapeutic strategy to enhance cancer immunotherapy.
Qingyu Lin, Tong Liu, Xingwen Wang, Guixue Hou, Zhiyuan Xiang, Wenxin Zhang, Shanliang Zheng, Dong Zhao, Qibin Leng, Xiaoshi Zhang, Minqiao Lu, Tianqi Guan, Hao Liu, Ying Hu
Despite advances in acute care, ischemic stroke remains a major cause of long-term disability. Approaches targeting both neuronal and glial responses are needed to enhance recovery and improve long-term outcome. The complement C3a receptor (C3aR) is a regulator of inflammation with roles in neurodevelopment, neural plasticity, and neurodegeneration. Using mice lacking C3aR (C3aR–/–) and mice overexpressing C3a in the brain, we uncovered two opposing effects of C3aR signaling on functional recovery after ischemic stroke: inhibition in the acute phase and facilitation in the later phase. Peri-infarct astrocyte reactivity was increased and density of microglia reduced in C3aR–/– mice, C3a overexpression led to the opposite effects. Pharmacological treatment of wild-type mice with intranasal C3a starting 7 days after stroke accelerated recovery of motor function and attenuated astrocyte reactivity without enhancing microgliosis. C3a treatment stimulated global white matter reorganization, increased peri-infarct structural connectivity and upregulated Igf1 and Thbs4 in the peri-infarct cortex. Thus, C3a treatment from day 7 after stroke exerts positive effects on astrocytes and neuronal connectivity while avoiding the deleterious consequences of C3aR signaling during the acute phase. Intranasal administration of C3aR agonists within convenient time window holds translational promise to improve outcome after ischemic stroke.
Anna Stokowska, Markus Aswendt, Daniel Zucha, Stephanie Lohmann, Frederique Wieters, Javier Moran Suarez, Alison L. Atkins, YiXian Li, Maria Miteva, Julia Lewin, Dirk Wiedermann, Michael Diedenhofen, Åsa Torinsson Naluai, Pavel Abaffy, Lukas Valihrach, Mikael Kubista, Mathias Hoehn, Milos Pekny, Marcela Pekna
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes from mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, the relationship between how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.
Alan J. Russell, Mike DuVall, Benjamin Barthel, Ying Qian, Angela K. Peter, Breanne L. Newell-Stamper, Kevin Hunt, Sarah J. Lehman, Molly R. Madden, Stephen T. Schlachter, Benjamin D. Robertson, Ashleigh Van Deusen, Hector M. Rodriguez, Carlos D. Vera, Yu Su, Dennis R. Claflin, Susan V. Brooks, Peter P. Nghiem, Alexis Rutledge, Twlya I. Juehne, Jinsheng Yu, Elisabeth R. Barton, Yangyi E. Luo, Andreas Patsalos, Laszlo Nagy, H. Lee Sweeney, Leslie A. Leinwand, Kevin Koch
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair proteins in 20-25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-containing chemotherapy. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1 and BRCA2) and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling, leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to block tumor growth while simultaneously stimulating an immune response.
Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel E. Hernandez, Amanda Ackermann, Sarina Porcella, Despoina Kalfakakou, Igor Dolgalev, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call CerTra syndrome. These findings uncover a central role for CERT autoregulation in the control of the sphingolipid biosynthetic flux, provide unexpected insight into the structural organisation of CERT, and suggest a possible therapeutic approach for CerTra syndrome patients.
Charlotte Gehin, Museer A. Lone, Winston Lee, Laura Capolupo, Sylvia Ho, Adekemi M. Adeyemi, Erica H. Gerkes, Alexander P.A. Stegmann, Estrella López-Martín, Eva Bermejo-Sánchez, Beatriz Martínez-Delgado, Christiane Zweier, Cornelia Kraus, Bernt Popp, Vincent Strehlow, Daniel Gräfe, Ina Knerr, Eppie R. Jones, Stefano Zamuner, Luciano A. Abriata, Vidya Kunnathully, Brandon E. Moeller, Anthony Vocat, Samuel Rommelaere, Jean-Philippe Bocquete, Evelyne Ruchti, Greta Limoni, Marine Van Campenhoudt, Samuel Bourgeat, Petra Henklein, Christian Gilissen, Bregje W. van Bon, Rolph Pfundt, Marjolein H. Willemsen, Jolanda H. Schieving, Emanuela Leonardi, Fiorenza Soli, Alessandra Murgia, Hui Guo, Qiumeng Zhang, Kun Xia, Christina R. Fagerberg, Christoph P. Beier, Martin J. Larsen, Irene Valenzuela, Paula Fernández-Álvarez, Shiyi Xiong, Robert Śmigiel, Vanesa López-González, Lluís Armengol, Manuela Morleo, Angelo Selicorni, Annalaura Torella, Moira Blyth, Nicola S. Cooper, Valerie Wilson, Renske Oegema, Yvan Herenger, Aurore Garde, Ange-Line Bruel, Frederic Tran Mau-Them, Alexis B.R. Maddocks, Jennifer M. Bain, Musadiq A. Bhat, Gregory Costain, Peter Kannu, Ashish Marwaha, Neena L. Champaigne, Michael J. Friez, Ellen B. Richardson, Vykuntaraju K. Gowda, Varunvenkat M. Srinivasan, Yask Gupta, Tze Y. Lim, Simone Sanna-Cherchi, Bruno Lemaitre, Toshiyuki Yamaji, Kentaro Hanada, John E. Burke, Ana Marija Jakšić, Brian D. McCabe, Paolo De Los Rios, Thorsten Hornemann, Giovanni D'Angelo, Vincenzo A Gennarino
STAT2 is a transcription factor activated by type I and III interferons. We report 23 patients with loss of function variants causing autosomal recessive (AR), complete STAT2 deficiency. Both cells transfected with mutant STAT2 alleles and the patients’ cells display impaired expression of interferon stimulated genes and impaired control of in-vitro viral infections. Clinical manifestations from early childhood onward include severe adverse reaction to live attenuated viral vaccines (LAV, 12/17 patients) and severe viral infections (10/23 patients), particularly critical influenza pneumonia (6 patients), critical COVID-19 pneumonia (1 patient), and herpes simplex encephalitis (1 patient). The patients display various types of hyperinflammation, often triggered by viral infection or after LAV administration, which probably attests to unresolved viral infection in the absence of STAT2-dependent type I and III IFN immunity (7 patients). Transcriptomic analysis reveals that circulating monocytes, neutrophils, and CD8 memory T cells contribute to this inflammation. Eight patients died (35%, 2 months-7 years): one of HSV-1 encephalitis, one of fulminant hepatitis, and six of heart failure during a febrile illness with no identified etiology. 15 patients remain alive (5-40 years). AR complete STAT2 deficiency underlies severe viral diseases, with half of the patients surviving into teenage years or adulthood.
Giorgia Bucciol, Leen Moens, Masato Ogishi, Darawan Rinchai, Daniela Matuozzo, Mana Momenilandi, Nacim Kerrouche, Catherine M. Cale, Elsa R. Treffeisen, Mohammad Al Salamah, Bandar K. Al-Saud, Alain Lachaux, Remi Duclaux-Loras, Marie Meignien, Aziz Bousfiha, Ibtihal Benhsaien, Anna Shcherbina, Anna Roppelt, Florian Gothe, Nadhira Houhou-Fidouh, Scott J. Hackett, Lisa M. Bartnikas, Michelle C. Maciag, Mohammed F. Alosaimi, Janet Chou, Reem W. Mohammed, Bishara J. Freij, Emmanuelle Jouanguy, Shen-Ying Zhang, Stephanie Boisson-Dupuis, Vivien Béziat, Qian Zhang, Christopher J.A. Duncan, Sophie Hambleton, Jean-Laurent Casanova, Isabelle Meyts
BACKGROUND. Lung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CFTR modulators improve activity of dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections. METHODS. We performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI) on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR and sequencing. RESULTS. Mean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Achromobacter and Burkholderia spp. decreased by 2-3 log10 CFU/ml after 1 month of ETI. However, most participants remained culture-positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture-negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture-negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria. CONCLUSIONS. Treatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment. TRIAL REGISTRATION. The trial registered at www.ClinicalTrials.gov as NCT04038047. FUNDING. This study was funded by the Cystic Fibtosis Foundation (PROMISE-MICRO18K1 and SINGH19R0) and NIH (R01HL148274).
David P. Nichols, Sarah J. Morgan, Michelle Skalland, Anh T. Vo, Jill M. Van Dalfsen, Sachinkumar B.P. Singh, Wendy Ni, Lucas R. Hoffman, Kailee McGeer, Sonya L. Heltshe, John P. Clancy, Steven M. Rowe, Peter K. Jorth, Pradeep K. Singh
Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay and subsequent intellectual disability, secondary microcephaly, and epilepsy. Preclinical studies evaluated an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50. In vitro studies demonstrated that transduction of patient-derived fibroblasts with AAV2/AP4M1 resulted in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1 knockout mice were intrathecally (IT) injected with 5E11, 2.5E11, or 1.25E11 vg doses of AAV9/AP4M1 at postnatal day p7-10 (pre-manifesting cohorts) or p90 (early manifesting cohorts). Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in wild-type mice, rats, and non-human primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1E15 vg. Of note, similar degrees of minimal to mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future IT AAV studies. These preclinical results identify an acceptably safe and efficacious dose of IT-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.
Xin Chen, Thomas Dong, Yuhui Hu, Raffaella De Pace, Rafael Mattera, Kathrin Eberhardt, Marvin Ziegler, Terry Pirovolakis, Mustafa Sahin, Juan S. Bonifacino, Darius Ebrahimi-Fakhari, Steven J. Gray
BACKGROUND. Refractory CMV viremia and disease are associated with significant morbidity and mortality in recipients of hematopoietic stem cell transplant (HCT). METHODS. In Phase I/II trials, we treated 67 subjects for CMV viremia or disease arising after allogeneic hematopoietic cell transplant with adoptive transfer of banked off-the-shelf, 3rd party, CMVpp65-sensitized T cells (CMVpp65-VSTs). All were evaluable for toxicity and 59 for response. Evaluable subjects had CMV disease or persisting viremia that had failed at least two weeks of induction therapy with a median of 3 antiviral drugs; 84.7% had >3/11 high risk features. CMVpp65-VSTs were specific for 1-3 CMVpp65 epitopes, presented by a limited set of HLA class I or II alleles, and were selected based on high resolution HLA matching at 2/10 HLA alleles and matching for subject and subject’s HCT donor for ≥1 allele through which the CMVpp65-VSTs were restricted. RESULTS. T-cell infusions were well tolerated. Of 59 subjects evaluable for response, 38 (64%) achieved complete or durable partial responses. CONCLUSIONS. Recipients responding to CMVpp65VSTs experienced an improved overall survival. Of the risk factors evaluated, transplant type, recipient CD4+ and CD8+ T-cell levels prior to adoptive therapy, and the HLA-restriction of CMVpp65-VSTs infused each significantly affected responses. In addition, CMVpp65-specific T cells of HCT donor or recipient origin contribute to the durability of both complete and partial responses. TRIAL REGISTRATION. The trials describe were registered with the NIH as follows: NCT00674648, NCT01646645 and NCT02136797. They were single center investigator-initiated trials and were not industry sponsored. FUNDING. This study was supported by funding from the National Institute of Health (P01 CA23766, R21 CA162002 and P30 CA008748), the Aubrey Fund, Claire Tow Foundation, Major Family Foundation, “Rick” Eisemann Pediatric Research Fund, Banbury Foundation, Edith Robertson Foundation, and Larry Smead Foundation.
Susan E. Prockop, Aisha N. Hasan, Ekaterina Doubrovina, Parastoo B. Dahi, M. Irene Rodriguez-Sanchez, Michael Curry, Audrey Mauguen, Genovefa A. Papanicolaou, Yiqi Su, JinJuan Yao, Maria E. Arcila, Farid Boulad, Hugo Castro-Malaspina, Christina Cho, Kevin J. Curran, Sergio Giralt, Nancy A Kernan, Guenther Koehne, Ann Jakubowski, Esperanza Papadopoulos, Miguel-Angel Perales, Ioannis Politikos, Keith J. Price, Annamalai Selvakumar, Craig S. Sauter, Roni Tamari, Teresa Vizconde, James W. Young, Richard J. O'Reilly
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