There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
Kun Ding, Jikui Shen, Zibran Hafiz, Sean F. Hackett, Raquel Lima e Silva, Mahmood Khan, Valeria E. Lorenc, Daiqin Chen, Rishi Chadha, Minie Zhang, Sherri Van Everen, Nicholas Buss, Michele Fiscella, Olivier Danos, Peter A. Campochiaro
Checkpoint blockade antibodies have been approved as immunotherapy for multiple types of cancer, but the response rate and efficacy are still limited. There are few immunogenic cell death (ICD)-inducing drugs available that can kill cancer cells, enhance tumor immunogenicity, increase the in vivo immune infiltration, and thereby boosting a tumor response to immunotherapy. So far, the ICD markers have been identified as the few immuno-stimulating characteristics of dead cells, but whether the presence of such ICD markers on tumor cells translates into enhanced antitumor immunity in vivo is still investigational. To identify anticancer drugs that could induce tumor cell death and boost T cell response, we performed drug screenings based on both an ICD reporter assay and T cell activation assay. We identified that teniposide, a DNA topoisomerase II inhibitor, could induce high mobility group box 1 (HMGB1) release and type I interferon signaling in tumor cells, and teniposide-treated tumor cells could activate antitumor T cell response both in vitro and in vivo. Mechanistically, teniposide induced tumor cell DNA damage and innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the activation of dendritic cells and subsequent T cells. Furthermore, teniposide potentiated the antitumor efficacy of anti-PD1 on multiple types of mouse tumor models. Our findings showed that teniposide could trigger tumor immunogenicity, and enabled a potential chemo-immunotherapeutic approach to potentiate the therapeutic efficacy of anti-PD1 immunotherapy.
Zining Wang, Jiemin Chen, Jie Hu, Hongxia Zhang, Feifei Xu, Wenzhuo He, Xiaojuan Wang, Mengyun Li, Wenhua Lu, Gucheng Zeng, Penghui Zhou, Peng Huang, Siyu Chen, Wende Li, Liang-ping Xia, Xiaojun Xia
T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.
Max Rieckmann, Murilo Delgobo, Chiara Gaal, Lotte Büchner, Philipp Steinau, Dan Reshef, Cristina Gil-Cruz, Ellis N. ter Horst, Malte Kircher, Theresa Reiter, Katrin G. Heinze, Hans W.M. Niessen, Paul A.J. Krijnen, Anja M. van der Laan, Jan J. Piek, Charlotte Koch, Hans-Jürgen Wester, Constantin Lapa, Wolfgang R. Bauer, Burkhard Ludewig, Nir Friedman, Stefan Frantz, Ulrich Hofmann, Gustavo Campos Ramos
A vaccine for hepatitis C virus (HCV) is urgently needed. Development of broadly-neutralizing plasma antibodies during acute infection is associated with HCV clearance, but the viral epitopes of these plasma antibodies are unknown. Identification of these epitopes could define the specificity and function of neutralizing antibodies (NAbs) that should be induced by a vaccine. Here, we present development and application of a high-throughput method that deconvolutes polyclonal anti-HCV NAbs in plasma, delineating the epitope specificities of anti-HCV NAbs in acute infection plasma of forty-four humans with subsequent clearance or persistence of HCV. Remarkably, we identified multiple broadly neutralizing antibody (bNAb) combinations that were associated with greater plasma neutralizing breadth and with HCV clearance. These studies have potential to inform new strategies for vaccine development by identifying bNAb combinations in plasma associated with natural clearance of HCV, while also providing a high-throughput assay that could identify these responses after vaccination trials.
Valerie J. Kinchen, Guido Massaccesi, Andrew I. Flyak, Madeleine C. Mankowski, Michelle D. Colbert, William O. Osburn, Stuart C. Ray, Andrea L. Cox, James E. Crowe Jr., Justin R. Bailey
Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.
Deepak Kumar, Manasi Das, Consuelo Sauceda, Lesley G. Ellies, Karina Kuo, Purva Parwal, Mehak Kaur, Lily Jih, Gautam K. Bandyopadhyay, Douglas Burton, Rohit Loomba, Olivia Osborn, Nicholas J.G. Webster
Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.
W. Brian Dalton, Eric Helmenstine, Noel Walsh, Lukasz P. Gondek, Dhanashree S. Kelkar, Abigail Read, Rachael Natrajan, Eric S. Christenson, Barbara Roman, Samarjit Das, Liang Zhao, Robert D. Leone, Daniel Shinn, Taylor Groginski, Anil K. Madugundu, Arun Patil, Daniel J. Zabransky, Arielle Medford, Justin Lee, Alex J. Cole, Marc Rosen, Maya Thakar, Alexander Ambinder, Joshua Donaldson, Amy E. DeZern, Karen Cravero, David Chu, Rafael Madero-Marroquin, Akhilesh Pandey, Paula J. Hurley, Josh Lauring, Ben Park
Histone H3K27 demethylase, JMJD3 plays a critical role in gene expression and T-cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here we show that JMJD3 deficiency in CD4+ T cells resulted in an accumulation of T cells in the thymus, and reduction of T cell number in the secondary lymphoid organs. We identified PDLIM4 as a significantly down-regulated target gene in JMJD3-deficient CD4+ T cells by gene profiling and ChIP-seq analyses. We further showed that PDLIM4 functioned as an adaptor protein to interact with S1P1 and filamentous actin (F-actin), thus serving as a key regulator of T cell trafficking. Mechanistically, JMJD3 bound to the promoter and gene body regions of Pdlim4 gene and regulated its expression by interacting with zinc finger transcription factor KLF2. Our findings have identified Pdlim4 as a JMJD3 target gene that affects T-cell trafficking by cooperating with S1P1, and provided insights into the molecular mechanisms by which JMJD3 regulates genes involved in T cell trafficking.
Chuntang Fu, Qingtian Li, Jia Zou, Changsheng Xing, Mei Luo, Bingnan Yin, Junjun Chu, Jiaming Yu, Xin Liu, Helen Y. Wang, Rong-Fu Wang
Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole genome RNA sequencing, gene set enrichment analysis and immunohistochemistry. Our analyses revealed five mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: (i) AR-high tumors (ARPC), (ii) AR-low tumors (ARLPC), (iii) amphicrine tumors composed of cells co-expressing AR and NE genes (AMPC), (iv) double-negative tumors (i.e. AR-/NE-; DNPC) and (v) tumors with small cell or NE gene expression without AR activity (SCNPC). RE1-silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the five mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.
Mark P. Labrecque, Ilsa M. Coleman, Lisha G. Brown, Lawrence D. True, Lori Kollath, Bryce Lakely, Holly M. Nguyen, Yu C. Yang, Rui M. Gil da Costa, Arja Kaipainen, Roger Coleman, Celestia S. Higano, Evan Y. Yu, Heather H. Cheng, Elahe A. Mostaghel, Bruce Montgomery, Michael T. Schweizer, Andrew C. Hsieh, Daniel W. Lin, Eva Corey, Peter S. Nelson, Colm Morrissey
The rate of disease progression in autosomal-dominant (AD) polycystic kidney disease (PKD) exhibits high intra-familial variability suggesting that environmental factors may play a role. We hypothesized that a prevalent form of renal insult may accelerate cystic progression and investigated tubular crystal deposition. We report that calcium oxalate (CaOx) crystal deposition led to rapid tubule dilation, activation of PKD-associated signaling pathways, and hypertrophy in tubule segments along the affected nephrons. Blocking mTOR signaling blunted this response and inhibited efficient excretion of lodged crystals. This mechanism of “flushing out” crystals by purposefully dilating renal tubules has not previously been recognized. Challenging PKD rat models with CaOx crystal deposition, or inducing calcium phosphate deposition by increasing dietary phosphorous intake, led to increased cystogenesis and disease progression. In a cohort of ADPKD patients, lower levels of urinary excretion of citrate, an endogenous inhibitor of calcium crystal formation, correlated with increased disease severity. These results suggest that PKD progression may be accelerated by commonly occurring renal crystal deposition which could be therapeutically controlled by relatively simple measures.
Jacob A. Torres, Mina Rezaei, Caroline Broderick, Louis Lin, Xiaofang Wang, Bernd Hoppe, Benjamin D. Cowley, Jr., Vincenzo Savica, Vicente E. Torres, Saeed Khan, Ross P. Holmes, Michal Mrug, Thomas Weimbs
To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.
William R. McManus, Michael J. Bale, Jonathan Spindler, Ann Wiegand, Andrew Musick, Sean C. Patro, Michele D. Sobolewski, Victoria K. Musick, Elizabeth M. Anderson, Joshua C. Cyktor, Elias K. Halvas, Wei Shao, Daria Wells, Xiaolin Wu, Brandon F. Keele, Jeffrey M. Milush, Rebecca Hoh, John W. Mellors, Stephen H. Hughes, Steven G. Deeks, John M. Coffin, Mary F. Kearney
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