Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally-induced seizures, corrected background electrocorticography activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof-of-concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.
Saja Fadila, Bertrand Beucher, Iria González Dopeso-Reyes, Anat Mavashov, Marina Brusel, Karen Anderson, Caroline Ismeurt, Ethan M. Goldberg, Ana Ricobaraza, Ruben Hernandez-Alcoceba, Eric J. Kremer, Moran Rubinstein
BACKGROUND. Adoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications. METHODS. We provide results of a personalized T-cell manufacturing program evaluating donor, patient, T-cell product and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T-lymphocyte (EBV-CTL) products from stem cell donors (SCD), related third party donors (TPD) or unrelated TPD from the allogeneic T-cell donor registry (alloCELL) established at Hannover Medical School were manufactured by immunomagnetic selection using CliniMACS Plus or Prodigy device and EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated and patient outcome and side effects were retrieved by retrospective chart analysis. RESULTS. Forty clinical-grade EBV-CTL products from SCDs, related or unrelated TPDs were generated for 37 patients with and without transplantation (Tx) history within 5 days (median) after donor identification. 34 patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients’ blood were detectable in 16/18 monitored patients (89 %) after transfer and correlated with clinical response. CONCLUSION. In conclusion, personalized clinical-grade manufacturing of EBV-CTL products via immunomagnetic selection from SCD, related or unrelated TPD is feasible in a timely manner. Overall, EBV-CTL were clinically effective and well-tolerated. Our data suggest EBV-CTL as promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT as well as patients with pre-existing organ dysfunction. TRIAL REGISTRATION. Not applicable. FUNDING. This study was in part funded by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), the Wilhelm-Sander-Stiftung (http://www.wilhelm-sander-stiftung.de, 2015.097.1), the Ellen-Schmidt-Program of the Hannover Medical School, and the German Federal Ministry of Education and Research (reference number: 01EO0802).
Agnes Bonifacius, Britta Lamottke, Sabine Tischer-Zimmermann, Rebecca Schultze-Florey, Lilia Goudeva, Hans-Gert Heuft, Lubomir Arseniev, Rita Beier, Gernot Beutel, Gunnar Cario, Birgit Fröhlich, Johann Greil, Leo Hansmann, Justin Hasenkamp, Michaela Höfs, Patrick Hundsdoerfer, Edgar Jost, Kinan Kafa, Oliver Kriege, Nicolaus Kröger, Stephan Mathas, Roland Meisel, Michaela Nathrath, Mervi Putkonen, Sarina Ravens, Hans Christian Reinhardt, Elisa Sala, Martin G. Sauer, Clemens Schmitt, Roland Schroers, Nina Kristin Steckel, Ralf Ulrich Trappe, Mareike Verbeek, Daniel Wolff, Rainer Blasczyk, Britta Eiz-Vesper, Britta Maecker-Kolhoff
Out-of-hospital cardiac arrest is a leading cause of death in the US, with a mortality rate over 90%. Preclinical studies demonstrate that cooling during cardiopulmonary resuscitation (CPR) is highly beneficial, but can be challenging to implement clinically. No medications exist for improving long-term cardiac arrest survival. We have developed a 20–amino acid peptide, TAT-PHLPP9c, that mimics cooling protection by enhancing AKT activation via PH domain leucine-rich repeat phosphatase 1 (PHLPP1) inhibition. Complementary studies were conducted in mouse and swine. C57BL/6 mice were randomized into blinded saline control and peptide-treatment groups. Following a 12-minute asystolic arrest, TAT-PHLPP9c was administered intravenously during CPR and significantly improved the return of spontaneous circulation, mean arterial blood pressure and cerebral blood flow, cardiac and neurological function, and survival (4 hour and 5 day). It inhibited PHLPP-NHERF1 binding, enhanced AKT but not PKC phosphorylation, decreased pyruvate dehydrogenase phosphorylation and sorbitol production, and increased ATP generation in heart and brain. TAT-PHLPP9c treatment also reduced plasma taurine and glutamate concentrations after resuscitation. The protective benefit of TAT-PHLPP9c was validated in a swine cardiac arrest model of ventricular fibrillation. In conclusion, TAT-PHLPP9c may improve neurologically intact cardiac arrest survival without the need for physical cooling.
Jing Li, Xiangdong Zhu, Matt T. Oberdier, Chunpei Lee, Shaoxia Lin, Sarah J. Fink, Cody N. Justice, Kevin Qin, Andrew W. Begeman, Frederick C. Damen, Hajwa Kim, Jiwang Chen, Kejia Cai, Henry R. Halperin, Terry L. Vanden Hoek
Although a disease-modifying therapy for CLN2 disease now exists, a poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2R207X mice, which carry one of the most common pathogenic mutations in human patients, but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities including spontaneous seizures, providing a robust and quantifiable disease-relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord, and differs markedly from the staging seen in mouse models of other forms of NCL. Neonatal administration of adeno-associated virus 9 (AAV9)-mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the lifespan of Cln2R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging pre-clinical efficacy of therapeutic interventions for CLN2 disease.
Keigo Takahashi, Elizabeth M. Eultgen, Sophie H. Wang, Nicholas R. Rensing, Hemanth R. Nelvagal, Joshua T. Dearborn, Olivier Danos, Nicholas Buss, Mark S. Sands, Michael Wong, Jonathan D. Cooper
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
The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer’s disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau transgenic mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.
Ha-Lim Song, Na-Young Kim, Jaewan Park, Meong Il Kim, Yu-Na Jeon, Se-Jong Lee, Kwangmin Cho, Young-Lim Shim, Kyoung-Hye Lee, Yeon-Seon Mun, Jung-A Song, Min-Seok Kim, Chan-Gi Pack, Minkyo Jung, Hyemin Jang, Duk L. Na, Minsun Hong, Dong-Hou Kim, Seung-Yong Yoon
Gary J. Macfarlane, Marcus Beasley, Gareth T. Jones
Lorenza Bellusci, Hana Golding, Surender Khurana
Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we show that deleting c-Src abrogates the activity of Forkhead Box M1 (FOXM1), a master transcriptional regulator of the cell cycle, in a genetically engineered model mimicking the Luminal B molecular subtype of breast cancer. By phosphorylating it on two tyrosine residues, c-Src stimulates the nuclear localization of FOXM1 and the expression of its target genes, including key regulators of G2-M cell cycle progression as well as c-Src itself. This positive feedback loop drives proliferation in genetically engineered and patient-derived models of Luminal B-like breast cancer. Targeting this mechanism, including through novel compounds that destabilize the FOXM1 protein, induces G2-M cell cycle arrest and apoptosis, blocking tumor progression and impairing metastasis. We identify a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the Luminal B subtype, which responds poorly to approved therapies. These findings indicate that a regulatory network centered on c-Src and FOXM1 is a targetable vulnerability in aggressive luminal breast cancers.
Ipshita Nandi, Harvey W. Smith, Virginie Sanguin-Gendreau, Linjia Ji, Alain Pacis, Vasilios Papavasiliou, Dongmei Zuo, Stella Nam, Sherif S. Attalla, Sung Hoon Kim, Sierra Lusson, Hellen Kuasne, Anne-Marie Fortier, Paul Savage, Constanza Martinez Ramirez, Morag Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen, William J. Muller
Bruton’s tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.
Vivian Changying Jiang, Yang Liu, Junwei Lian, Shengjian Huang, Alexa Jordan, Qingsong Cai, Ruitao Lin, Fangfang Yan, Joseph McIntosh, Yijing Li, Yuxuan Che, Zhihong Chen, Jovanny Vargas, Maria Badillo, John Nelson Bigcal, Heng-Huan Lee, Wei Wang, Yixin Yao, Lei Nie, Christopher R. Flowers, Michael Wang
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