Person: Balaj, Leonora
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Balaj
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Leonora
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Balaj, Leonora
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Publication Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles(Nature Publishing Group UK, 2018) Reátegui, Eduardo; van der Vos, Kristan E.; Lai, Charles P.; Zeinali, Mahnaz; Atai, Nadia; Aldikacti, Berent; Floyd, Frederick P.; H. Khankhel, Aimal; Thapar, Vishal; Hochberg, Fred H.; Sequist, Lecia; Nahed, Brian; S. Carter, Bob; Toner, Mehmet; Balaj, Leonora; T. Ting, David; Breakefield, Xandra; Stott, ShannonExtracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform (EVHB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EVHB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per μL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods. Our approach allows for the subsequent release of captured tumor EVs, enabling downstream characterization and functional studies. Processing serum and plasma samples from glioblastoma multiforme (GBM) patients, we can detect the mutant EGFRvIII mRNA. Moreover, using next-generation RNA sequencing, we identify genes specific to GBM as well as transcripts that are hallmarks for the four genetic subtypes of the disease.Publication Dopa-Responsive Dystonia: Functional Analysis of Single Nucleotide Substitutions within the 5’ Untranslated GCH1 Region(Public Library of Science, 2013) Armata, Ioanna A.; Balaj, Leonora; Kuster, John K.; Zhang, Xuan; Tsai, Shelun; Armatas, Andreas A.; Multhaupt-Buell, Trisha J.; Soberman, Roy; Breakefield, Xandra; Ichinose, Hiroshi; Sharma, NutanBackground: Mutations in the GCH1 gene are associated with childhood onset, dopa-responsive dystonia (DRD). Correct diagnosis of DRD is crucial, given the potential for complete recovery once treated with L-dopa. The majority of DRD associated mutations lie within the coding region of the GCH1 gene, but three additional single nucleotide sequence substitutions have been reported within the 5’ untranslated (5’UTR) region of the mRNA. The biologic significance of these 5’UTR GCH1 sequence substitutions has not been analyzed. Methodology/Principal Findings Luciferase reporter assays, quantitative real time PCR and RNA decay assays, combined with bioinformatics, revealed a pathogenic 5’UTR GCH1 substitution. The +142C>T single nucleotide 5’UTR substitution that segregates with affected status in DRD patients, substantially attenuates translation without altering RNA expression levels or stability. The +142C>T substitution disrupts translation most likely by creating an upstream initiation start codon (uAUG) and an upstream open reading frame (uORF). Conclusions/Significance: This is the first GCH1 regulatory substitution reported to act at a post-transcriptional level, increasing the list of genetic diseases caused by abnormal translation and reaffirming the importance of investigating potential regulatory substitutions in genetic diseases.Publication BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles(Nature Publishing Group, 2013) Chen, Walter; Balaj, Leonora; Liau, Linda M; Samuels, Michael L; Kotsopoulos, Steve K; Maguire, Casey; LoGuidice, Lori; Soto, Horacio; Garrett, Matthew; Zhu, Lin Dan; Sivaraman, Sarada; Chen, Clark; Wong, Eric T; Carter, Bob S; Hochberg, Fred H; Breakefield, Xandra; Skog, JohanDevelopment 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.Publication Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma(Nature Pub. Group, 2015) Shao, Huilin; Chung, Jaehoon; Lee, Kyungheon; Balaj, Leonora; Min, Changwook; Carter, Bob S.; Hochberg, Fred H.; Breakefield, Xandra; Lee, Hakho; Weissleder, RalphReal-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.Publication Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium(Co-Action Publishing, 2015) Quesenberry, Peter J.; Aliotta, Jason; Camussi, Giovanni; Abdel-Mageed, Asim B.; Wen, Sicheng; Goldberg, Laura; Zhang, Huang-Ge; Tetta, Ciro; Franklin, Jeffrey; Coffey, Robert J.; Danielson, Kirsty; Subramanya, Vinita; Ghiran, Ionita; Das, Saumya; Chen, Clark C.; Pusic, Kae M.; Pusic, Aya D.; Chatterjee, Devasis; Kraig, Richard P.; Balaj, Leonora; Dooner, MarkThe NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.Publication Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium(Co-Action Publishing, 2015) Laurent, Louise C.; Abdel-Mageed, Asim B.; Adelson, P. David; Arango, Jorge; Balaj, Leonora; Breakefield, Xandra; Carlson, Elizabeth; Carter, Bob S.; Majem, Blanca; Chen, Clark C.; Cocucci, Emanuele; Danielson, Kirsty; Courtright, Amanda; Das, Saumya; Elmageed, Zakaria Y. Abd; Enderle, Daniel; Ezrin, Alan; Ferrer, Marc; Freedman, Jane; Galas, David; Gandhi, Roopali; Huentelman, Matthew J.; Van Keuren-Jensen, Kendall; Kalani, Yashar; Kim, Yong; Krichevsky, Anna; Lai, Charles; Lal-Nag, Madhu; Laurent, Clara D.; Leonardo, Trevor; Li, Feng; Malenica, Ivana; Mondal, Debasis; Nejad, Parham; Patel, Tushar; Raffai, Robert L.; Rubio, Renee; Skog, Johan; Spetzler, Robert; Sun, Jie; Tanriverdi, Kahraman; Vickers, Kasey; Wang, Liang; Wang, Yaoyu; Wei, Zhiyun; Weiner, Howard; Wong, David; Yan, Irene K.; Yeri, Ashish; Gould, StephenExtracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.Publication Heparin affinity purification of extracellular vesicles(Nature Publishing Group, 2015) Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos; Breakefield, Xandra; Skog, Johan; Maguire, CaseyExtracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.Publication A High-Throughput Magneto-Electrochemical Array for the Integrated Isolation and Profiling of Extracellular Vesicles From Plasma(2021-06-28) Park, Jongmin; Park, Jun Seok; Huang, Chen-han; Jo, Ala; Cook, Kaitlyn; Wang, Rui; Lin, Hsing-Ying; Van Deun, Jan; Li, Huiyan; Min, Jouha; Wang, Lan; Yoon, Ghilsuk; Carter, Bob; Balaj, Leonora; Choi, Gyu-Seog; Castro, Cesar; Weissleder, Ralph; Lee, HakhoPublication Coding and noncoding landscape of extracellular RNA released by human glioma stem cells(Nature Publishing Group UK, 2017) Wei, Zhiyun; Batagov, Arsen O.; Schinelli, Sergio; Wang, Jintu; Wang, Yang; El Fatimy, Rachid; Rabinovsky, Rosalia; Balaj, Leonora; Chen, Clark C.; Hochberg, Fred; Carter, Bob; Breakefield, Xandra; Krichevsky, AnnaTumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.Publication Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles(American Association for the Advancement of Science, 2018) Ricklefs, Franz L.; Alayo, Quazim; Krenzlin, Harald; Mahmoud, Ahmad; Speranza, Maria C.; Nakashima, Hiroshi; Hayes, Josie L.; Lee, Kyungheon; Balaj, Leonora; Passaro, Carmela; Rooj, Arun; Krasemann, Susanne; Carter, Bob; Chen, Clark C.; Steed, Tyler; Treiber, Jeffrey; Rodig, Scott; Yang, Katherine; Nakano, Ichiro; Lee, Hakho; Weissleder, Ralph; Breakefield, Xandra; Godlewski, Jakub; Westphal, Manfred; Lamszus, Katrin; Freeman, Gordon; Bronisz, Agnieszka; Lawler, Sean; Chiocca, E.Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.