Exosomes are natural carriers of exogenous siRNA to human cells in vitro

© 2013 Shtam et al. Background: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies. Results: This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells. Conclusions: The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy. © 2013 Shtam et al.; licensee BioMed Central Ltd.Russian Federal Program “Scientific and Scientific-Pedagogical Personnel of Innovative Russia”, contract 14.740.11.0754 and Fellowship from the Administration of Leningrad region to Shtam T

Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p<0.001) that was confirmed by mode size detected by NTA (p<0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool

Therapeutic prospects of extracellular vesicles in cancer treatment

Extracellular vesicles (EVs) are released by all cells within the tumor microenvironment, such as endothelial cells, tumor-associated fibroblasts, pericytes and immune system cells. The EVs carry the cargo of parental cells formed of proteins and nucleic acids, which can convey cell-to-cell communication influencing the maintenance and spread of the malignant neoplasm, for example promoting angiogenesis, tumor cell invasion and immune escape. However, EVs can also suppress tumor progression, either by the direct influence of the protein and nucleic acid cargo of the EVs or via antigen presentation to immune cells as tumor derived EVs carry on their surface some of the same antigens as the donor cells. Moreover, dendritic cell-derived EVs carry MHC class I and class II/peptide complexes and are able to prime other immune system cell types and activate an anti-tumor immune response. Given the relative longevity of vesicles within the circulation and their ability to cross blood-brain barriers, modification of these unique organelles offers the potential to create new biological-tools for cancer therapy. This review examines how modification of the EV cargo has the potential to target specific tumor mechanisms responsible for tumor formation and progression to develop new therapeutic strategies and to increase the efficacy of antitumor therapies

EVALUATION OF EXPRESSION OF 4 MIRNAS IN CYTOLOGICAL SAMPLES AS AN ADDITIONAL METHOD OF CERVICAL CANCER DIAGNOSIS

Introduction. Cervical cancer is the 4th most common cancer among women. The main screening method for cervical cancer is cytological examination of the cervical epithelium. This method allows to evaluate the level of cervical dysplasia (malignant potential) but it has several limitations and flaws. Development and implementation of new methods of molecular and genetic analysis in clinical practice can increase informational value of the traditional cytological examination and therefore objectivity in choosing treatment options.Objective is to develop and verify a new method of differential diagnosis of severe intraepithelial dysplasia and invasive cervical cancer.Materials and methods. The method is based on analysis of small non-coding RNA molecules (miRNAs) extracted from the material of traditional Pap smears. Based on literature search, 18 “marker” microRNA molecules were chosen and their expression levels were estimated in 166 samples of Pap smears from cervical canals with different cytological diagnoses. The analysis was performed using reverse transcription polymerase chain reaction.Results. Estimation of ratios between expression levels of miRNA pairs: 126/375; 20а/375; 126/145 allows to differentiate with high confidence borderline states of severe intraepithelial dysplasia and invasive cervical carcinoma (coefficients of quantitative interpretation of the error curve were 0.8, 0.75, 0.72, respectively).Conclusions. Analysis of miRNAs in Pap smear samples is a promising additional method of cervical cancer diagnosis. The method is objective and can be proposed as a supporting technique in cases when cytological examination doesn’t allow to differentiate between borderline pathological states of the cervical epithelium. Implementation of the method in clinical practice requires methodological optimization and additional validation using more clinical material

КОМПЛАЕНС-КОНТРОЛЬ В БАНКОВСКОЙ ОРГАНИЗАЦИИ

The scientific article is devoted to the research analysis of the theoretical aspects of the concept of "compliance control" in the formation of the credit risk management policy of commercial banks of the Russian Federation. The relevance of the research is due to the growth trend in the volume of assets of the country's banking system, associated with the active process of lending to legal entities and individuals, which requires the improvement of the credit risk management system. The article examines the theoretical foundations of the economic essence of the concept of "compliance control". The factors and reasons that induce the formation of a compliance control system in domestic banks are highlighted. The process of forming a compliance control system in banking organizations is considered. Compliance control functions are listed.Научная статья посвящена исследовательскому анализу теоретических аспектов понятия «комплаенс-контроль» при формировании политики управления кредитными рисками коммерческих банков Российской Федерации. Актуальность научного исследования обусловлена тенденцией роста объема активов банковской системы страны, связанного с активным процессом кредитования юридических и физических лиц, что требует совершенствования системы управления кредитными рисками. В рамках статьи рассмотрены теоретические основы экономической сущности понятия «комплаенс-контроль». Выделены факторы и причины, побуждающие формирование системы комплаенсконтроля в отечественных банках. Рассмотрен процесс формирования системы комплаенс-контроля в банковских организациях. Перечислены функции комплаенс-контроля

Blood Plasma Exosomes Contain Circulating DNA in Their Crown

It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that exosomes from healthy females (HF) or breast cancer patients (BCP) plasma contain less than 1.4 &times; 10&minus;8 pg of DNA. Thus, only a minor part of cirDNA is attached to the outer side of the exosome as part of the vesicle crown: the share of exosomal DNA does not exceed 0.025% HF plasma DNA and 0.004% BCP plasma DNA. Treatment of plasma exosomes with DNase I with subsequent dot immunoassay reveals that H2a, H2b, and H3 histones are not part of the exosomal membrane, but are part of the cirDNA&ndash;protein macromolecular complex associated with the surface of the exosome either through interaction with DNA-binding proteins or with histone-binding proteins. Using bioinformatics approaches after identification by MALDI-TOF mass spectrometry, 16 exosomal DNA-binding proteins were identified. It was shown that four proteins&mdash;AIFM1, IGHM, CHD5, and KCNIP3&mdash;are candidates for DNA binding on the outer membrane of exosomes; the crown of exosomes may include five DNA-binding proteins: H2a, H2b, H3, IGHM, and ALB. Of note, AIFM1, IGHM, and CHD5 proteins are found only in HF plasma exosomes; KCNIP3 protein is identified only in BCP plasma exosomes; and H2a, H2b, H3, and ALB are revealed in all samples of plasma exosomes. Two histone-binding proteins, CHD5 and KDM6B, have been found in exosomes from HF plasma. The data obtained indicate that cirDNA preferentially binds to the outer membrane of exosomes by association with DNA-binding proteins

Abnormal activity of transcription factors gli in high-grade gliomas.

Malignant transformation is associated with loss of cell differentiation, anaplasia. Transcription factors gli, required for embryonic development, may be involved in this process. We studied the activity of transcription factors gli in high-grade gliomas and their role in maintenance of stem cell state and glioma cell survival. 20 glioma cell lines and a sample of a normal adult brain tissue were used in the present study. We found the expression of gli target genes, including GLI1 and FOXM1, in all tested glioma cell lines, but not in the normal tissue. Interestingly, the expression of gli target genes in some glioma cell lines was observed together with a high level of their transcriptional repressor, Gli3R. Knockdown of GLI3 in one of these lines resulted in decrease of gli target gene expression. These data suggest that Gli3R does not prevent the gli target genes transcription, and gli3 acts in glioma cells more as an activator, than a repressor of transcription. We observed that gli regulated the expression of such genes, as SOX2 or OCT4 that maintain stem cell state, and TET1, involving in DNA demethylation. Treatment with GANT61 or siRNA against GLI1, GLI2, or GLI3 could result in complete glioma cell death, while cyclopamine had a weaker and line-specific effect on glioma cell survival. Thus, the gli transcription factors are abnormally active in high-grade gliomas, regulate expression of genes, maintaining the stem cell state, and contribute to glioma cell survival

Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers

Extracellular vesicles (EV) are involved in important processes of glioblastoma multiforme (GBM), including malignancy and invasion. EV secreted by glioblastoma cells may cross the hematoencephalic barrier and carry molecular cargo derived from the tumor into the peripheral circulation. Therefore, the determination of the molecular composition of exosomes released by glioblastoma cells seems to be a promising approach for the development of non-invasive methods of the detection of the specific exosomal protein markers in the peripheral blood. The present study aimed to determine the common exosomal proteins presented in preparations from different cell lines and search potential glioblastoma biomarkers in exosomes. We have performed proteomics analysis of exosomes obtained from the conditioned culture medium of five glioblastoma cell lines. A list of 133 proteins common for all these samples was generated. Based on the data obtained, virtual two-dimensional electrophoresis (2DE) maps of proteins presented in exosomes of glioblastoma cells were constructed and the gene ontology (GO) analysis of exosome proteins was performed. A correlation between overexpressed in glial cell proteins and their presence in exosomes have been found. Thus, the existence of many potential glioblastoma biomarkers in exosomes was confirmed