43 results on '"Xandra O. Breakefield"'
Search Results
2. Heparin interferes with the uptake of liposomes in glioma
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Thomas S. van Solinge, Kristina Pagh Friis, Killian O'Brien, Romy L. Verschoor, Jeroen van Aarle, Arnold Koekman, Xandra O. Breakefield, Pieter Vader, Raymond Schiffelers, and Marike Broekman
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Liposomes ,Heparin ,Glioma ,Glioblastoma ,Uptake ,Delivery ,Pharmacy and materia medica ,RS1-441 - Abstract
In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only.
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- 2023
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3. Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2
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Shilpa Prabhakar, Roberta L. Beauchamp, Pike See Cheah, Akiko Yoshinaga, Edwina Abou Haidar, Sevda Lule, Gayathri Mani, Katia Maalouf, Anat Stemmer-Rachamimov, David H. Jung, D. Bradley Welling, Marco Giovannini, Scott R. Plotkin, Casey A. Maguire, Vijaya Ramesh, and Xandra O. Breakefield
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neurofibromatosis type 2 ,gene therapy ,adeno-associated viral vector ,schwannoma ,Schwann cells ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.
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- 2022
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4. CRISPR-Cas knockout of miR21 reduces glioma growth
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Lisa Nieland, Thomas S. van Solinge, Pike See Cheah, Liza M. Morsett, Joseph El Khoury, Joseph I. Rissman, Benjamin P. Kleinstiver, Marike L.D. Broekman, Xandra O. Breakefield, and Erik R. Abels
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glioblastoma ,CRISPR ,gene editing ,microRNA ,miR-21 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.
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- 2022
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5. Illuminating cellular and extracellular vesicle-mediated communication via a split-Nanoluc reporter in vitro and in vivo
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Thomas S. van Solinge, Shadi Mahjoum, Stefano Ughetto, Alessandro Sammarco, Marike L.D. Broekman, Xandra O. Breakefield, and Killian P. O’Brien
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CP: Cell biology ,Biotechnology ,TP248.13-248.65 ,Biochemistry ,QD415-436 ,Science - Abstract
Summary: Tools to effectively demonstrate and quantify functional delivery in cellular communication have been lacking. This study reports the use of a fluorescently labeled split Nanoluc reporter system to demonstrate and quantify functional transfer between cells in vitro and in a subcutaneous tumor mouse model. Our construct allows monitoring of direct, indirect, and specifically extracellular vesicle-mediated functional communication. Motivation: In tracking protein-mediated cellular communication, few tools distinguish between proteins that escape the endosome and are functional within the cytosol and proteins that are degraded by the lysosome or released back into the extracellular space. We designed a tool that signals functional protein delivery and can be used to study cell-to-cell and extracellular vesicle-mediated communication in vitro and in vivo.
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- 2023
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6. Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function
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Lilian Cruz, Bence György, Pike See Cheah, Benjamin P. Kleinstiver, William A. Eimer, Sara P. Garcia, Nutan Sharma, Laurie J. Ozelius, D. Cristopher Bragg, J. Keith Joung, Osmar Norberto de Souza, Luis Fernando Saraiva Macedo Timmers, and Xandra O. Breakefield
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CRISPR ,dystonia ,DYT1 ,torsinA ,herpes simplex virus type 1 ,TOR1A ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
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- 2020
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7. Long-Term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1
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Shilpa Prabhakar, Pike See Cheah, Xuan Zhang, Max Zinter, Maria Gianatasio, Eloise Hudry, Roderick T. Bronson, David J. Kwiatkowski, Anat Stemmer-Rachamimov, Casey A. Maguire, Miguel Sena-Esteves, Bakhos A. Tannous, and Xandra O. Breakefield
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Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mammalian target of rapamycin (mTOR)-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal nodules, subependymal giant cell astrocytomas, and cortical tubers in the human brain. Neurological manifestations in TSC include intellectual disability, autism, hydrocephalus, and epilepsy. In a stochastic mouse model of TSC1 brain lesions, complete loss of Tsc1 is achieved in homozygous Tsc1-floxed mice in a subpopulation of neural cells in the brain by intracerebroventricular (i.c.v.) injection at birth of an adeno-associated virus (AAV) vector encoding Cre recombinase. This results in median survival of 38 days and brain pathology, including subependymal lesions and enlargement of neuronal cells. Remarkably, when these mice were injected intravenously on day 21 with an AAV9 vector encoding hamartin, most survived at least up to 429 days in apparently healthy condition with marked reduction in brain pathology. Thus, a single intravenous administration of an AAV vector encoding hamartin restored protein function in enough cells in the brain to extend lifespan in this TSC1 mouse model. Keywords: brain, neurology, tumor suppressor, gene therapy
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- 2019
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8. Discovery and Verification of Extracellular miRNA Biomarkers for Non-invasive Prediction of Pre-eclampsia in Asymptomatic Women
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Srimeenakshi Srinivasan, Ryan Treacy, Tiffany Herrero, Richelle Olsen, Trevor R. Leonardo, Xuan Zhang, Peter DeHoff, Cuong To, Lara G. Poling, Aileen Fernando, Sandra Leon-Garcia, Katharine Knepper, Vy Tran, Morgan Meads, Jennifer Tasarz, Aishwarya Vuppala, Soojin Park, Clara D. Laurent, Tony Bui, Pike See Cheah, Rachael Tabitha Overcash, Gladys A. Ramos, Hilary Roeder, Ionita Ghiran, Mana Parast, Xandra O. Breakefield, Amir J. Lueth, Sharon R. Rust, Max T. Dufford, Angela C. Fox, Durlin E. Hickok, Julja Burchard, J. Jay Boniface, Louise C. Laurent, Kim A. Boggess, George R. Saade, Scott A. Sullivan, Glenn R. Markenson, Jay D. Iams, Dean V. Coonrod, Leonardo M. Pereira, M. Sean Esplin, Larry M. Cousins, Garrett K. Lam, and Matthew K. Hoffman
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pre-eclampsia ,biomarkers ,bivariate ,miRNA ,extracellular RNA ,small RNA-seq ,Medicine (General) ,R5-920 - Abstract
Summary: Development of effective prevention and treatment strategies for pre-eclampsia is limited by the lack of accurate methods for identification of at-risk pregnancies. We performed small RNA sequencing (RNA-seq) of maternal serum extracellular RNAs (exRNAs) to discover and verify microRNAs (miRNAs) differentially expressed in patients who later developed pre-eclampsia. Sera collected from 73 pre-eclampsia cases and 139 controls between 17 and 28 weeks gestational age (GA), divided into separate discovery and verification cohorts, are analyzed by small RNA-seq. Discovery and verification of univariate and bivariate miRNA biomarkers reveal that bivariate biomarkers verify at a markedly higher rate than univariate biomarkers. The majority of verified biomarkers contain miR-155-5p, which has been reported to mediate the pre-eclampsia-associated repression of endothelial nitric oxide synthase (eNOS) by tumor necrosis factor alpha (TNF-α). Deconvolution analysis reveals that several verified miRNA biomarkers come from the placenta and are likely carried by placenta-specific extracellular vesicles.
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- 2020
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9. Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells
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Rocco Lucero, Valentina Zappulli, Alessandro Sammarco, Oscar D. Murillo, Pike See Cheah, Srimeenakshi Srinivasan, Eric Tai, David T. Ting, Zhiyun Wei, Matthew E. Roth, Louise C. Laurent, Anna M. Krichevsky, Xandra O. Breakefield, and Aleksandar Milosavljevic
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Biology (General) ,QH301-705.5 - Abstract
Summary: Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers. : Extensive intercellular interactions occur within the notoriously heterogeneous tumor microenvironment of GBM. Lucero et al. identify distinct angiogenic gene regulatory responses of brain endothelial cells to growth factors and to extracellular vesicles (EVs) secreted by GBM stem-like cells. The response to EVs shows a footprint of EV-derived miRNAs. Keywords: angiogenesis, biomarker, cancer stem cell, deconvolution, glioblastoma, exRNA, extracellular vesicle, miRNA, reprogramming, tumor microenvironment
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- 2020
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10. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease
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Bence György, Camilla Lööv, Mikołaj P. Zaborowski, Shuko Takeda, Benjamin P. Kleinstiver, Caitlin Commins, Ksenia Kastanenka, Dakai Mu, Adrienn Volak, Vilmantas Giedraitis, Lars Lannfelt, Casey A. Maguire, J. Keith Joung, Bradley T. Hyman, Xandra O. Breakefield, and Martin Ingelsson
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Alzheimer's disease ,Swedish mutation ,adeno-associated virus ,genome editing ,CRISPR ,amyloid precursor protein ,amyloid-β ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.
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- 2018
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11. Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21
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Erik R. Abels, Sybren L.N. Maas, Lisa Nieland, Zhiyun Wei, Pike See Cheah, Eric Tai, Christy-Joy Kolsteeg, Sophie A. Dusoswa, David T. Ting, Suzanne Hickman, Joseph El Khoury, Anna M. Krichevsky, Marike L.D. Broekman, and Xandra O. Breakefield
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Biology (General) ,QH301-705.5 - Abstract
Summary: Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression. : Abels et al. show miR-21 transfer from glioma to microglia by palmitoylated GFP-labeled extracellular vesicles in vivo. This transfer results in miR-21 target-specific mRNA downregulation. Following downregulation of Btg2, proliferation in microglia is increased, suggesting reprogramming of microglia in the tumor microenvironment through extracellular vesicles shed by glioma cells.
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- 2019
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12. Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles
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Mikołaj Piotr Zaborowski, Kyungheon Lee, Young Jeong Na, Alessandro Sammarco, Xuan Zhang, Marcin Iwanicki, Pike See Cheah, Hsing-Ying Lin, Max Zinter, Chung-Yu Chou, Giulia Fulci, Bakhos A. Tannous, Charles Pin-Kuang Lai, Michael J. Birrer, Ralph Weissleder, Hakho Lee, and Xandra O. Breakefield
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Biology (General) ,QH301-705.5 - Abstract
Summary: Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type. : Cancer cell-derived extracellular vesicles (EVs) can be used in diagnostics, but their enrichment remains challenging. Zaborowski et al. identify membrane proteins enriched on the surface of cancer cells compared with normal tissues using TCGA, the Human Protein Atlas, and GTEx and present methods to measure immunocapture of cancer EVs in vitro and in animal models. Keywords: extracellular vesicles, membrane proteins, biomarker, The Cancer Genome Atlas, Genotype-Tissue Expression Project, Human Protein Atlas, palmitoylated fluorescent protein, membrane-bound Gaussia luciferase
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- 2019
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13. Survival benefit and phenotypic improvement by hamartin gene therapy in a tuberous sclerosis mouse brain model
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Shilpa Prabhakar, Xuan Zhang, June Goto, Sangyeul Han, Charles Lai, Roderick Bronson, Miguel Sena-Esteves, Vijaya Ramesh, Anat Stemmer-Rachamimov, David J. Kwiatkowski, and Xandra O. Breakefield
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Tuberous sclerosis complex ,TSC ,TSC1 ,TSC2 ,Gene therapy ,AAV ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1cc (homozygous for a conditional floxed Tsc1 allele) SynI-cre+, in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1ccSynIcre+ mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients.
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- 2015
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14. Molecular pathways in dystonia
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D. Cristopher Bragg, Ioanna A. Armata, Flavia C. Nery, Xandra O. Breakefield, and Nutan Sharma
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Dystonia ,TorsinA ,THAP1 ,PACT ,PRKRA ,Dopamine ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The hereditary dystonias comprise a set of diseases defined by a common constellation of motor deficits. These disorders are most likely associated with different molecular etiologies, many of which have yet to be elucidated. Here we discuss recent advances in three forms of hereditary dystonia, DYT1, DYT6 and DYT16, which share a similar clinical picture: onset in childhood or adolescence, progressive spread of symptoms with generalized involvement of body regions and a steady state affliction without treatment. Unlike DYT1, the genes responsible for DYT6 and DYT16 have only recently been identified, with relatively little information about the function of the encoded proteins. Nevertheless, recent data suggest that these proteins may fit together within interacting pathways involved in dopaminergic signaling, transcriptional regulation, and cellular stress responses. This review focuses on these molecular pathways, highlighting potential common themes among these dystonias which may serve as areas for future research. This article is part of a Special Issue entitled “Advances in dystonia”.
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- 2011
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15. Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics
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Jeffrey W. Hewett, Juan Zeng, Brian P. Niland, D. Cristopher Bragg, and Xandra O. Breakefield
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Intermediate filaments ,Dystonia ,DYT1 ,Vimentin ,Fibroblasts ,Cell adhesion ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms—recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA—the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.
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- 2006
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16. A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe
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Shanta M. Messerli, Shilpa Prabhakar, Yi Tang, Khalid Shah, Maria L. Cortes, Vidya Murthy, Ralph Weissleder, Xandra O. Breakefield, and Ching-Hsuan Tung
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Caspase-1 ,near-infrared fluorescence (NIRF) ,apoptosis ,brain tumors ,HSV ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin β-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-IacZ) expressing a catalytically active caspase-1-IacZ fusion protein; 3) inhibition of HGC-ICE-IacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.
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- 2004
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17. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part II. Vector Systems and Applications
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Andreas Jacobs, Xandra O. Breakefield, and Cornel Fraefel
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glioma ,gene therapy ,recombinant HSV-1 ,amplicon ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Many properties of HSV-1 are especially suitable for using this virus as a vector to treat diseases affecting the central nervous system (CNS), such as Parkinson's disease or malignant gliomas. These advantageous properties include natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. Selective oncolysis in combination with modulation of the immune response mediated by replication-conditional HSV-1 vectors appears to be a highly promising approach in the battle against malignant glioma. Helper virus-free HSV/AAV hybrid amplicon vectors have great promise in mediating long-term gene expression in the PNS and CNS for the treatment of various neurodegenerative disorders or chronic pain. Current research focuses on the design of HSV-1-derived vectors which are targeted to certain cell types and support transcriptionally regulatable transgene expression. Here, we review the recent developments on HSV-1-based vector systems and their applications in experimental and clinical gene therapy protocols.
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- 1999
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18. HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis
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Andreas Jacobs, Xandra O. Breakefield, and Cornel Fraefel
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herpes simplex virus ,gene therapy ,recombinant HSV-1 ,amplicon ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1) virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.
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- 1999
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19. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression
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Andreas Jacobs, Michael Dubrovin, Jeff Hewett, Miguel Sena-Esteves, Cui-Wen Tan, Mark Slack, Michele Sadelain, Xandra O. Breakefield, and Juri G. Tjuvajev
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thymidine kinase ,ganciclovir ,FIAU ,cancer gene therapy ,fusion genes ,imaging ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.
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- 1999
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20. Contributors
- Author
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Samira Abdulai-Saiku, Stanley H. Appel, Arthur P. Arnold, Lisa M. Arnold, Robert M. Arnold, Alexa Bacha, Miroslav 'Misha' Backonja, Zinzi D. Bailey, Lucinda Bateman, David R. Beers, Anna Berti, Mamta Bhatnagar, Devin K. Binder, Marina Boido, Maura Boldrini, David Borsook, Xandra O. Breakefield, Robert H. Brown, Rami Burstein, Eduardo R. Butelman, Louis R. Caplan, S. Chen, Marie-Françoise Chesselet, Stefan Clemens, Paula R. Clemens, Joseph T. Coyle, John C. DeWitt, Dena B. Dubal, Veljko Dubljević, Eva L. Feldman, Beth A. Fischer, M.C. Flux, J.S. Fortin, Angelisa Frasca, Francesca Garbarini, Thomas Gasser, Charles F. Gillespie, Michael S. Gold, Stefan M. Gold, Randi Hagerman, Regan Hamel, Craig Haney, James C. Harris, Sara Hassani, Norman J. Haughey, Vibol Heng, J. Horn, Rosana-Bristena Ionescu, Raffaele Iorio, David J. Irwin, Henry J. Kaminski, Dalia Khammash, Vikram Khurana, Charlotte Kilstrup-Nielsen, Bhumsoo Kim, Boram Kim, Marieke Klein, Nastassja Koen, Glenn T. Konopaske, Joanna A. Korecka, Birgitte Rahbek Kornum, Mary Jeanne Kreek, Krister Kristensson, Grzegorz Krzak, Linda L. Kusner, Nicoletta Landsberger, Edward B. Lee, Tong Li, Paweł P. Liberski, Christine Lochner, Christopher A. Lowry, J. John Mann, Clara Marincowitz, E.A. Mayer, E.D. Mayer, Iris Coates McCall, Louise D. McCullough, Michael J. Meaney, Claudio Melo de Gusmao, Abhishek L. Menesgere, Emmanuel Mignot, William C. Mobley, Mayra Montalvo, Alisha R. Moreland-Capuia, Marco Neppi-Modona, Alexandra M. Nicaise, Rae Nishi, Orna O'Toole, Cassia Overk, Laurie Ozelius, Matthew P. Parsons, H.B. Penticoff, Luca Peruzzotti-Jametti, Owen M. Peters, Allison Peterson, Jessica M. Phan, Sean J. Pittock, Stefano Pluchino, Thad A. Polk, Araya Puwanant, Shreya K. Rajagopal, Vijayalakshmi Ravindranath, Lynn A. Raymond, Brian Reed, Kerry J. Ressler, Diane L. Ritchie, Leah H. Rubin, Stacey A. Sakowski, Mario A. Saporta, Alena V. Savonenko, Helen E. Scharfman, Bruce K. Shapiro, Nutan Sharma, Cayce K. Shaw, Michael E. Shy, Beata Sikorska, Ethan J. Silverman, Roger P. Simon, Kristina Simonyan, Catrina Sims-Robinson, Richard Jay Smeyne, Clay Smith, Colin Smith, Sharan R. Srinivasan, Dan J. Stein, Christopher D. Stephen, Indu Subramanian, Edina Szabo, Alissa A. Thomas, Luis B. Tovar-y-Romo, Arshya Vahabzadeh, Alessandro Vercelli, Ashley Viera-Ortiz, Mitchell T. Wallin, Donna M. Werling, Thomas Wichmann, Clayton A. Wiley, David R. Williams, Cory Willis, Philip C. Wong, Vadim Yuferov, Weihua Zhao, Michael J. Zigmond, and Saša A. Živković
- Published
- 2023
21. Dystonia
- Author
-
Christopher D. Stephen, Kristina Simonyan, Laurie Ozelius, Xandra O. Breakefield, and Nutan Sharma
- Published
- 2023
22. Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics
- Author
-
Xandra O. Breakefield, Juan Zeng, Jeffrey W. Hewett, Brian P. Niland, and D. Cristopher Bragg
- Subjects
Cytoplasm ,Dystonia Musculorum Deformans ,Cytoplasmic Streaming ,Kinesins ,Vimentin ,DYT1 ,macromolecular substances ,Microtubules ,Cell Line ,lcsh:RC321-571 ,Motor protein ,Tubulin ,Cell Line, Tumor ,Neurites ,Humans ,Intermediate filaments ,Cell adhesion ,Intermediate filament ,Cytoskeleton ,education ,Cell Shape ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Cells, Cultured ,Actin ,education.field_of_study ,biology ,HSC70 Heat-Shock Proteins ,Brain ,Fibroblasts ,Molecular biology ,Actins ,Cell biology ,Dystonia ,Neurology ,Kinesin light chain 1 ,Mutation ,biology.protein ,Kinesin ,Microtubule-Associated Proteins ,Molecular Chaperones - Abstract
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.
- Published
- 2006
23. Detection of Spontaneous Schwannomas by MRI in a Transgenic Murine Model of Neurofibromatosis Type 2
- Author
-
Roderick T. Bronson, Yi Tang, Marco Giovannini, Ralph Weissleder, Shanta M. Messerli, and Xandra O. Breakefield
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,tumor ,neurofibromatosis ,medicine.diagnostic_test ,H&E stain ,Schwann cell ,Magnetic resonance imaging ,Biology ,Schwannoma ,transgenic mice ,medicine.disease ,herpes simplex virus ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,lcsh:RC254-282 ,Merlin (protein) ,medicine.anatomical_structure ,medicine ,otorhinolaryngologic diseases ,Immunohistochemistry ,magnetic resonance imaging ,Neurofibromatosis ,Neurofibromatosis type 2 - Abstract
Spontaneous schwannomas were detected by magnetic resonance imaging (MRI) in a transgenic murine model of neurofibromatosis type 2 (NF2) expressing a dominant mutant form of merlin under the Schwann cell-specific PO promoter. Approximately 85% of the investigated mice showed putative tumors by 24 months of age. Specifically, 21% of the mice showed tumors in the intercostal muscles, 14% in the limb muscles, 7% in the spinal cord and spinal ganglia, 7% in the external ear, 14% in the muscle of the abdominal region, and 7% in the intestine; 66% of the female mice had uterine tumors. Multiple tumors were detected by MRI in 21% of mice. The tumors were isointense with muscle by T1-weighted MRI, showed strong enhancement following administration of gadolinium-DTPA, and were markedly hyperintense by T2-weighted MRI, all hallmarks of the clinical manifestation. Hematoxylin and eosin staining and immunohistochemistry indicated that the tumors consisted of schwannomas and Schwann cell hyperplasias. The lesions stained positively for S-100 protein and a marker antigen for the mutated transgenic NF2 protein, confirming that the imaged tumors and areas of hyperplasia were of Schwann cell origin and expressed the mutated NF2 protein. Tumors were highly infectable with a recombinant herpes simplex virus type 1 vector, hrR3, which contains the reporter gene, lacZ. The ability to develop schwannoma growth with a noninvasive imaging technique will allow assessment of therapeutic interventions.
- Published
- 2002
24. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression
- Author
-
Xandra O. Breakefield, Juri Gelovani Tjuvajev, Michael Dubrovin, Mark Slack, Cui Wen Tan, Andreas Jacobs, Jeff Hewett, Michele Sadelain, and Miguel Sena-Esteves
- Subjects
Cancer Research ,DNA, Complementary ,ganciclovir ,Transgene ,Recombinant Fusion Proteins ,Blotting, Western ,Green Fluorescent Proteins ,cancer gene therapy ,Cell Separation ,Herpesvirus 1, Human ,Biology ,Marker gene ,Antiviral Agents ,lcsh:RC254-282 ,Green fluorescent protein ,Fusion gene ,Gene product ,Transduction, Genetic ,thymidine kinase ,Tumor Cells, Cultured ,Animals ,Transgenes ,Cloning, Molecular ,Promoter Regions, Genetic ,fusion genes ,Dose-Response Relationship, Drug ,Arabinofuranosyluracil ,imaging ,Genetic Therapy ,Cell sorting ,Flow Cytometry ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Molecular biology ,Fusion protein ,Rats ,Luminescent Proteins ,Retroviridae ,FIAU ,Microscopy, Fluorescence ,Thymidine kinase ,Research Article - Abstract
Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo . We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1- tk ) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1- tk genes was generated ( tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1- tk -specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.
- Published
- 1999
25. miR-1289 and 'Zipcode'-like Sequence Enrich mRNAs in Microvesicles
- Author
-
Sibylle Madlener, Xandra O. Breakefield, Erdogan Pekcan Erkan, Thomas Ströbel, Mehmet Fatih Bolukbasi, Jian-Bing Fan, Gokhan Baris Ozdener, Okay Saydam, and Arda Mizrak
- Subjects
Cell type ,Biology ,Bioinformatics ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,microRNA ,medicine ,Nucleotide ,miR-1289 ,030304 developmental biology ,Sequence (medicine) ,chemistry.chemical_classification ,0303 health sciences ,Messenger RNA ,lcsh:RM1-950 ,medicine.disease ,Microvesicles ,Genetic Materials ,Cell biology ,lcsh:Therapeutics. Pharmacology ,chemistry ,030220 oncology & carcinogenesis ,zipcode ,miRNAs ,Molecular Medicine ,Original Article ,microvesicles ,Glioblastoma - Abstract
Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.
- Published
- 2012
26. Contributors
- Author
-
Paul D. Acton, Kaveh Asadi-Moghaddam, Krystof Bankiewicz, Gerard J. Boer, Nicholas M. Boulis, Xandra O. Breakefield, Peter Carmeliet, E. Antonio Chiocca, Ronald G. Crystal, Jane Dunning, Matthew J. During, Marina E. Emborg, Thais Federici, David J. Fink, Helen L. Fitzsimons, John R. Forsayeth, Cornel Fraefel, Justin F. Fraser, Guangping Gao, Joseph C. Glorioso, Steven A. Goldman, Thomas A. Green, Neil R. Hackett, Piotr Hadaczek, William T.J. Hendriks, Charles E. Inturrisi, Luc Jasmin, Stephen M. Kaminsky, Michael G. Kaplitt, Matthias Klugmann, Diether Lambrechts, Patricia A. Lawlor, Claudia B. Leichtlein, Neal Luther, Marina Mata, Jerry R. Mendell, Anne Messer, Andra Miller, Todd W. Miller, Jeffrey Moirano, Sergei Musatov, Eric J. Nestler, Francesco Noé, Peter T. O'Hara, Sonoko Ogawa, Donald W. Pfaff, Harish Poptani, Marc J. Ruitenberg, Claudia Senn, Fraser Sim, Dolan Sondhi, Mark M. Souweidane, Qingshan Teng, Luk H. Vandenberghe, Joost Verhaagen, Annamaria Vezzani, Charles H. Vite, James M. Wilson, and John Wolfe
- Published
- 2006
27. HSV Amplicon Vectors for Gene Delivery to the Nervous System
- Author
-
Claudia Senn, Cornel Fraefel, and Xandra O. Breakefield
- Subjects
Retrovirus ,Transgene ,Neurotropism ,DNA replication ,Computational biology ,Biology ,Amplicon ,Gene delivery ,biology.organism_classification ,Fusion protein ,Virology ,Virus - Abstract
The HSV amplicon vector incorporates features of HSV-1, including a 150 kb transgene capacity, a viral origin of DNA replication and packaging signal, and virion proteins. The large transgene capacity is one of the most distinguishing features of this vector system, which allows incorporation of multiple transgenes and large genomic fragments, as well as informational elements from other virus vectors, including AAV, EBV, and retrovirus. Vectors have been modified to include elements, which increase infection of specific cell types and allow retention of transgene sequences, either as replicating episomal elements or through site-specific integration into the cell genome, and provide the ability to control transgene expression. The virion itself includes proteins that can be used to track infection and deliver fusion proteins to cells. Within the nervous system, these vectors are especially useful due to the natural neurotropism of the virus, with a strong retrograde component, and minimal perturbation of neuronal physiology. Vectors have been used to deliver proteins to facilitate fluorescence, bioluminescent, and magnetic resonance imaging, as well as to monitor neuronal functions in animal models involving learning/memory and addiction paradigms. Vectors have been designed to ameliorate symptoms in models of neurologic disease, including protection of neurons from toxic insults and replacement of genetically deficient proteins, as well as in treatment of brain tumors. Amplicon vectors are considered highly compatible with clinical trials due to their intrinsic lack of toxicity, but methods of production need to be improved to generate high titers and clinically compatible vector stocks.
- Published
- 2006
28. DYT1 Transgenic Mouse
- Author
-
Jeremy Petravicz, David G. Standaert, D. Cristopher Bragg, Xandra O. Breakefield, and Nutan Sharma
- Subjects
Dystonia ,Genetically modified mouse ,Torsion dystonia ,Mutation ,medicine ,Focal dystonia ,Biology ,medicine.disease ,medicine.disease_cause ,Motor learning ,Gene ,Neuroscience ,Motor skill - Abstract
Acquired focal dystonia can be precipitated by either extended practice of skilled movements or damage to the neuronal circuits that are implicated in motor learning. From these observations, investigators propose a theory that dystonia results from a failure of adaptive mechanisms that normally support the acquisition of new skills. This theory is supported by the demonstration of abnormalities in motor sequence learning in non-manifesting human carriers of the early-onset torsion dystonia gene (DYT1) mutation. The most common type of early-onset, generalized dystonia is a GAG deletion in the DYT1 (TOR1A) gene encoding torsin A. This mutation results in loss of a glutamic acid residue near the carboxy terminus of torsin A. Thus, it is reasonable to study mice carrying the DYT1 mutation for abnormalities in motor learning. The most widely accepted and validated tool for the study of motor skills in the mouse is the rotarod. Thus, in addition to systematically evaluating posture with an emphasis on detecting abnormal postures similar to those seen in humans with dystonia, investigators can study future generations of DYT1 transgenic mice for their ability to maintain balance and remain coordinated with repeated rotarod testing.
- Published
- 2005
29. List of Contributors
- Author
-
Tetsuo Ashizawa, P.C. Baier, Lore Becker, David J. Bennett, Brett Berke, Ranjita Betarbet, Kailash P. Bhatia, Francesco Bibbiani, David T. Blake, David R. Borchelt, Prodip Bose, D. Cristopher Bragg, Allison Brashear, Xandra O. Breakefield, Susan Bressman, Kathleen Burke, Nancy N. Byl, Guy A. Caldwell, Kim A. Caldwell, Songsong Cao, M. Angela Cenci, Marie-Françoise Chesselet, Carlo Colosimo, Mai Dang, Julie A. Dennis, Didier Devys, Paula Dietrich, Ioannis Dragatsis, Ian D'Souza, Rodger J. Elble, Craig Evinger, Pierre-Olivier Fernagut, Hubert H. Fernandez, John K. Fink, Sheila M. Fleming, Colin F. Fletcher, Lyle Fox, Stephen C. Fowler, Joseph H. Friedman, Felix Geser, Imad Ghorayeb, Monica Gorassini, J. Timothy Greenamyre, Philip J. Harvey, Simon J.R. Heales, Peter J. Healy, Dominique Helmlinger, Ellen J. Hess, Ralph Hillman, Gregg E. Homanics, Keith Hyland, Iyare Izevbaye, Vernice Jackson-Lewis, H.A. Jinnah, Anita J. Jurkowski, Iris S. Kassem, Michael D. Kaytor, Jason E. Kralic, Mark LeDoux, Jada Lewis, Yuqing Li, David Lieberman, Gary S. Linn, Irene Litvan, Paul J. Lombroso, Elan D. Louis, Martin Lundblad, J. Lawrence Marsh, Eileen McGowan, T.L. McKerchar, Michael Merzenich, A. Leslie Morrow, Robert Naquet, Richard Nass, Parvoneh Poorkaj Navas, Todd K. O'Buckley, Justin D. Oh, Chihiro Ohye, Harry T. Orr, Jessica L. Osterman, Leo J. Pallanck, Massimo Pandolfo, Robert G. Pendleton, Haixiang Peng, I-Feng Peng, Dianne M. Perez, Susan L. Perlman, Joel S. Perlmutter, Jeremy Petravicz, Ronald F. Pfeiffer, James O. Phillips, Michael R. Pranzatelli, Stefan-M. Pulst, Shirley Rainier, Jayaraman Rao, Angelika Richter, Farrel R. Robinson, Christopher A. Ross, Perminder S. Sachdev, Rachel Saunders-Pullman, Gerard D. Schellenberg, Gabriele Schilling, Peter R. Schofield, Nutan Sharma, Todd B. Sherer, Carmen Silva-Barrat, Harvey S. Singer, Richard Jay Smeyne, Constance Smith-Hicks, Mark Stacy, Nadia Stafanova, David G. Standaert, S.H. Subramony, Samer D. Tabbal, Kwok-Keung Tai, Floyd J. Thompson, Leslie M. Thompson, François Tison, Claudia Trenkwalder, Daniel D. Truong, Atsushi Ueda, Suvi Vartiainen, Hans Weiher, Avery H. Weiss, Gregor Karl Wenning, Alexander J. Whitworth, Peter A. Windsor, Garry Wong, Chun-Fang Wu, and T.J. Zarcone
- Published
- 2005
30. [34] HSV-1 amplicon vectors
- Author
-
Sam Wang, Xandra O. Breakefield, and Cornel Fraefel
- Subjects
HSL and HSV ,Amplicon ,Biology ,Virology - Published
- 2002
31. Herpes simplex virus type 1-based amplicon vector systems
- Author
-
David R. Jacoby, Cornel Fraefel, and Xandra O. Breakefield
- Subjects
Genetics ,viruses ,Transgene ,DNA replication ,Amplicon ,Biology ,medicine.disease_cause ,Genome ,Virology ,Plasmid ,Herpes simplex virus ,Lytic cycle ,medicine ,Gene - Abstract
Publisher Summary This chapter discusses the development of the herpes simplex virus type 1 (HSV-1) amplicon as an efficient gene transfer vehicle, with emphasis on recent advances that have reduced toxic effects associated with the vector and increased both the efficiency of gene transfer and the stability of gene expression. HSV-1-based amplicon vectors have a great potential for applications in gene therapy, because they have a large transgene capacity and can efficiently transduce most cell types, both dividing and nondividing. The HSV-1 amplicon contains three types of genetic elements—namely, (1) sequences that allow its propagation as a bacterial plasmid, (2) a transgene cassette with the genes of interest, and (3) ∼1% of the 152-kb HSV-1 genome, in particular an origin of DNA replication and a DNA cleavage/packaging signal. These two noncoding, cis-acting HSV-1 elements are sufficient for supporting the replication of amplicon DNA and subsequent packaging into HSV-1 virions in the presence of helper functions. HSV-1, a member of the subfamily Alphaherpesvirinae , is a common pathogen in humans and one of the most extensively characterized viruses. The lytic cycle of the HSV-1 infection usually causes only mild symptoms, such as cold sores, and is rapidly controlled by the immune system.
- Published
- 2000
32. Genetic Engineering for CNS Regeneration
- Author
-
Xandra O. Breakefield, Andreas Jacobs, and Sam Wang
- Subjects
Programmed cell death ,Neurite ,viruses ,Cell ,Biology ,Gene delivery ,Suicide gene ,medicine.disease_cause ,biology.organism_classification ,Virology ,Cell biology ,Herpes simplex virus ,medicine.anatomical_structure ,Lentivirus ,medicine ,Gene - Abstract
Publisher Summary There is a rich array of possible intervention points and times in stimulation of central nervous system (CNS) repair using genetic engineering techniques. These can be broken down into three cell categories: neurons, pathways, and innervation targets—and three time intervals—immediately after injury, during neurite regrowth, upon target recognition, and upon reinnervation. At the time of injury it should be possible to inject vectors and cells directly into the damaged site. The most nontoxic, yet efficient direct gene delivery vectors in the current formulations would be adenovirus and adenoassociated virus (AAV), gutless adenovirus, helper virus-free herpes simplex virus (HSV) amplicons, and lentivirus. Genes that might be helpful for neurons in the injury period include those coding for anti-apoptotic proteins or protective proteins for free radicals. Grafted cells could be preprogrammed genetically with a pro-drug-activation suicide gene, so that systemic application of the pro-drug would lead to cell death. An ideal system would be activation of a cell death geneunder the control of a tetracycline inducible promoter.
- Published
- 1999
33. Chapter 7 Analysis of MAOA mutations in humans
- Author
-
Christo Shalish, Xandra O. Breakefield, Yun-Pung P. Hsu, Dennis L. Murphy, Elizabeth A. Tivol, and Deborah E. Schuback
- Subjects
Genetics ,Mutation ,Exon ,Point mutation ,Haplotype ,medicine ,Single-strand conformation polymorphism ,Biology ,medicine.disease_cause ,Gene ,Phenotype ,Stop codon - Abstract
Publisher Summary This chapter presents an analysis of monoamine oxidase (MAO) A mutations in humans. A direct correlation between an MAOA mutation and a phenotype has been provided by recent studies of a pedigree in the Netherlands. Eight males in the pedigree, who are mildly retarded and show episodic aggressive behavior, including attempted murder, attempted rape, exhibitionism, attempted suicide, and arson, were found to carry a point mutation that converts a glutamine codon into a stop codon in the eighth exon. This mutation completely disrupts MAO-A activity. The common feature shared by these males of an apparent episodic loss of impulse control suggests the involvement of physiological changes resulting from MAO-A deficiency, but could also be related to mild retardation. The correlations of sequence variations with disease phenotypes, as in the MAO-A-deficient patients, may begin to emerge by the use of methods based on polymerase chain reaction (PCR) and single-strand conformational polymorphisms (SSCP). In addition, the allelic-association approach will continue to provide an efficient means for evaluating the role of MAO genes in different disease states.
- Published
- 1995
34. Gene delivery to the nervous system using retroviral vectors
- Author
-
Manish K. Aghi, Miguel Sena-Esteves, Edward M. Kaye, Xandra O. Breakefield, and Peter Pechan
- Subjects
biology ,viruses ,Cell ,Gene delivery ,biology.organism_classification ,Molecular biology ,Virus ,Antisense RNA ,Retrovirus ,medicine.anatomical_structure ,Viral envelope ,Viral entry ,Murine leukemia virus ,medicine - Abstract
Publisher Summary Retrovirus vectors derived from Moloney murine leukemia virus (MMLV) have been the most utilized vector for gene delivery to the nervous system. Particles enter the cells by binding to glycoproteins on the plasma membrane of the host cell, followed by fusion of the virus envelope with the cell membrane. There is some cell specificity of virus entry conferred by the interaction between envelope elements in the virus particles and the cell membrane. It is shown that stable, albeit low level, transgene expression could be achieved following peripheral grafting of fibroblasts by using the “house-keeping” promoter for dihydrofolate reductase, which is active in most mammalian cell types. The function of particular neural proteins is elucidated by retrovirus-encoded antisense RNA, which interferes with expression of the protein. Antisense technology is still an incomplete art and one can expect to achieve only partial inhibition of translation of the targeted mRNA. Retrovirus-mediated gene transfer is a particularly applicable technique for brain tumors because of the selectivity of retroviruses for proliferating cells. The possibility of obtaining retroviruses with mixed phenotypes, through infection of the same cell by different viruses, from the same or different taxonomic groups is also elaborated.
- Published
- 1995
35. GENETIC LINKAGE ANALYSIS IN FAMILIAL DYSAUTONOMIA USING A DNA PROBE FOR THE β-NERVE GROWTH FACTOR GENE
- Author
-
Xandra O. Breakefield, Gregory Orloff, Carmela M. Castiglione, Felicia B. Axelrod, Lisa Coussens, and Axel Ullrich
- Subjects
Genetics ,Nerve growth factor ,Familial dysautonomia ,Genetic linkage analysis ,medicine ,A-DNA ,Biology ,medicine.disease ,Molecular biology ,Gene - Published
- 1983
36. Catecholamine Metabolism and Drug Effects in Cultured Mammalian Cells: Analysis by HPLC
- Author
-
Peter A. Crooks, Xandra O. Breakefield, and James K. Coward
- Subjects
Drug ,Catecholamine metabolism ,Biochemistry ,Chemistry ,media_common.quotation_subject ,Pharmacology ,High-performance liquid chromatography ,media_common - Published
- 1979
37. MONOAMINE OXIDASE ACTIVITY IN FIBROBLASTS FROM CONTROL INDIVIDUALS AND PATIENTS WITH INHERITED NEUROLOGIC DISEASES
- Author
-
Maria R. Castro Costa, Carmela M. Castiglione, Susan B. Edelstein, and Xandra O. Breakefield
- Subjects
Dystonia ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Cultured skin ,business.industry ,Monoamine oxidase ,Duchenne muscular dystrophy ,medicine.disease ,Endocrinology ,Physical medicine and rehabilitation ,Familial dysautonomia ,Internal medicine ,medicine ,Muscular dystrophy ,business ,Normal range - Abstract
Monoamine oxidase (MAO) activity was measured in cultured skin fibroblasts from control individuals and patients with the Lesch-Nyhan syndrome, dystonia musculoram deformans, Duchenne muscular dystrophy and familial dysautonomia. The distribution of activity in the control population appeared to be bimodal; activities in the Lesch-Nyhan, dystonia and muscular dystrophy populations tended to be within the low normal range.
- Published
- 1979
38. CYCLIC GMP LEVELS IN NEUROBLASTOMA CELLS ARE INCREASED BY DEPOLARIZATION AND BY MUSCARINIC RECEPTOR ACTIVATION
- Author
-
Robert E. Study, Tamas Bartfai, Paul Greengard, and Xandra O. Breakefield
- Subjects
Neuroblastoma cell ,Cyclic gmp ,Chemistry ,Muscarinic acetylcholine receptor ,Depolarization ,Muscarinic acetylcholine receptor M1 ,Cell biology - Published
- 1978
39. CURRENT GENETIC APPROACHES TO THE MAMMALIAN NERVOUS SYSTEM
- Author
-
Michael B. Rosenberg, John E. Pintar, and Xandra O. Breakefield
- Subjects
Nervous system ,Zoology ,Computational biology ,Biology ,chemistry.chemical_compound ,Protein structure ,medicine.anatomical_structure ,chemistry ,Cell culture ,Genotype ,medicine ,Gene ,Developmental biology ,DNA ,Function (biology) - Abstract
Publisher Summary This chapter describes genetics as a set of concepts that provide unique insights into complex natural phenomena. It presents genetic concepts that are combined with new techniques in molecular biology, cell culture, biochemistry, and developmental biology to expand an understanding of the nervous system. Knowledge of the number, nature, and position of genes controlling neural properties explains the molecular basis of the expression, structure, function, and interaction of these properties. It is also possible to selectively alter the genotype of cells and to use these cells to create animals with known genetic lesions. Further, naturally occurring variations in DNA and protein structure can be assessed and the effects of these variations on neural function evaluated.
- Published
- 1982
40. MOLECULAR PROPERTIES OF PLATELET MAO IN PSYCHIATRIC PATIENTS AND CONTROLS
- Author
-
J. Wojciechowski, Carmela M. Castiglione, E. Giller, and Xandra O. Breakefield
- Subjects
Gel electrophoresis ,chemistry.chemical_classification ,medicine.medical_specialty ,biology ,Monoamine oxidase ,Pharmacology ,Tyramine ,Enzyme structure ,Enzyme assay ,chemistry.chemical_compound ,Enzyme ,chemistry ,medicine ,biology.protein ,Platelet ,Psychiatry ,Hormone - Abstract
Abnormal activities of platelet monoamine oxidase (MAO) have been reported in a number of neuropsychiatric disorders. All reports, however, are not consistent. Many factors may contribute to the variance in the measurement of MAO activity. These include the number of enzyme molecules, enzyme structure, platelet isolation procedure, enzyme activity assay procedure, drugs, hormones, activators and inhibitors. It may be necessary to evaluate other parameters of MAO in addition to enzyme activity. The finding of a lowered Vmax and Km for tyramine in chronic schizophrenic patients is reviewed. Procedures are described for quantitating the number of active MAO molecules with 3 H-pargyline and resolving labeled molecules on the basis of net charge by gel electrophoresis. MAO from platelets isolated by the washout technique from controls and psychiatric patients are compared. Enzyme activity and the number of active enzyme molecules are significantly correlated (r = 0.94). Patients and controls were similiar. These procedures can help to determine whether alterations in platelet MAO activity are due to differences in the number of enzyme molecules or factors that modulate the activity of the enzyme molecule.
- Published
- 1982
41. TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion
- Author
-
Kevin Rostasy, Sarah J Augood, Jeffrey W Hewett, Joanne Chung-on Leung, Hikaru Sasaki, Laurie J Ozelius, Vijaya Ramesh, David G Standaert, Xandra O Breakefield, and John C Hedreen
- Subjects
Torsin ,Dystonia ,Human brain ,Substantia nigra ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.
- Published
- 2003
- Full Text
- View/download PDF
42. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
- Author
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Walter W Chen, Leonora Balaj, Linda M Liau, Michael L Samuels, Steve K Kotsopoulos, Casey A Maguire, Lori LoGuidice, Horacio Soto, Matthew Garrett, Lin Dan Zhu, Sarada Sivaraman, Clark Chen, Eric T Wong, Bob S Carter, Fred H Hochberg, Xandra O Breakefield, and Johan Skog
- Subjects
BEAMing PCR ,biomarkers ,cancer ,extracellular vesicles ,droplet digital PCR ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.
- Published
- 2013
- Full Text
- View/download PDF
43. miR-1289 and 'Zipcode'-like Sequence Enrich mRNAs in Microvesicles
- Author
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Mehmet Fatih Bolukbasi, Arda Mizrak, Gokhan Baris Ozdener, Sibylle Madlener, Thomas Ströbel, Erdogan Pekcan Erkan, Jian-Bing Fan, Xandra O Breakefield, and Okay Saydam
- Subjects
microvesicles ,miRNAs ,miR-1289 ,zipcode ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.
- Published
- 2012
- Full Text
- View/download PDF
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