Characterizing Aptamer Interaction with the Oncolytic Virus VV-GMCSF-Lact.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 µM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer-virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer-virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.

Transcriptome Changes in Glioma Cells upon Infection with the Oncolytic Virus VV-GMCSF-Lact.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Design of the New Closo-Dodecarborate-Containing Gemcitabine Analogue for the Albumin-Based Theranostics Composition.

Combination therapy is becoming an increasingly important treatment strategy because multi-drugs can maximize therapeutic effect and overcome potential mechanisms of drug resistance. A new albumin-based theranostic containing gemcitabine closo-dodecaborate analogue has been developed for combining boron neutron capture therapy (BNCT) and chemotheraphy. An exo-heterocyclic amino group of gemcitabine was used to introduce closo-dodecaborate, and a 5'-hydroxy group was used to tether maleimide moiety through an acid-labile phosphamide linker. The N-trifluoroacylated homocysteine thiolactone was used to attach the gemcitabine analogue to human serum albumin (HSA) bearing Cy5 or Cy7 fluorescent dyes. The half-maximal inhibitory concentration (IC50) of the designed theranostic relative to T98G cells was 0.47 mM with the correlation coefficient R = 0.82. BNCT experiments resulted in a decrease in the viability of T98G cells, and the survival fraction was ≈ 0.4.

MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation.

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Transcriptome Changes in Glioma Cells Cultivated under Conditions of Neurosphere Formation.

Glioma is the most common and heterogeneous primary brain tumor. The development of a new relevant preclinical models is necessary. As research moves from cultures of adherent gliomas to a more relevant model, neurospheres, it is necessary to understand the changes that cells undergo at the transcriptome level. In the present work, we used three patient-derived gliomas and two immortalized glioblastomas, while their cultivation was carried out under adherent culture and neurosphere (NS) conditions. When comparing the transcriptomes of monolayer (ML) and NS cell cultures, we used Enrichr genes sets enrichment analysis to describe transcription factors (TFs) and the pathways involved in the formation of glioma NS. It was observed that NS formation is accompanied by the activation of five common gliomas of TFs, SOX2, UBTF, NFE2L2, TCF3 and STAT3. The sets of transcripts controlled by TFs MYC and MAX were suppressed in NS. Upregulated genes are involved in the processes of the epithelial-mesenchymal transition, cancer stemness, invasion and migration of glioma cells. However, MYC/MAX-dependent downregulated genes are involved in translation, focal adhesion and apical junction. Furthermore, we found three EGFR and FGFR signaling feedback regulators common to all analyzed gliomas-SPRY4, ERRFI1, and RAB31-which can be used for creating new therapeutic strategies of suppressing the invasion and progression of gliomas.

Aptamers for Addressed Boron Delivery in BNCT: Effect of Boron Cluster Attachment Site on Functional Activity.

Among the great variety of anti-cancer therapeutic strategies, boron neutron capture therapy (BNCT) represents a unique approach that doubles the targeting accuracy due to the precise positioning of a neutron beam and the addressed delivery of boron compounds. We have recently demonstrated the principal possibility of using a cell-specific 2'-F-RNA aptamer for the targeted delivery of boron clusters for BNCT. In the present study, we evaluated the amount of boron-loaded aptamer inside the cell via two independent methods: quantitative real-time polymerase chain reaction and inductive coupled plasma-atomic emission spectrometry. Both assays showed that the internalized boron level inside the cell exceeds 1 × 109 atoms/cell. We have synthesized closo-dodecaborate conjugates of 2'-F-RNA aptamers GL44 and Waz, with boron clusters attached either at the 3'- or at the 5'-end. The influence of cluster localization was evaluated in BNCT experiments on U-87 MG human glioblastoma cells and normal fibroblasts and subsequent analyses of cell viability via real-time cell monitoring and clonogenic assay. Both conjugates of GL44 aptamer provided a specific decrease in cell viability, while only the 3'-conjugate of the Waz aptamer showed the same effect. Thus, an individual adjustment of boron cluster localization is required for each aptamer. The efficacy of boron-loaded 2'-F-RNA conjugates was comparable to that of 10B-boronophenylalanine, so this type of boron delivery agent has good potential for BNCT due to such benefits as precise targeting, low toxicity and the possibility to use boron clusters made of natural, unenriched boron.

Aptamers Enhance Oncolytic Viruses' Antitumor Efficacy.

Oncolytic viruses are highly promising for cancer treatment because they target and lyse tumor cells. These genetically engineered vectors introduce therapeutic or immunostimulatory genes into the tumor. However, viral therapy is not always safe and effective. Several problems are related to oncolytic viruses' targeted delivery to the tumor and immune system neutralization in the bloodstream. Cryoprotection and preventing viral particles from aggregating during storage are other critical issues. Aptamers, short RNA, or DNA oligonucleotides may help to crawl through this bottleneck. They are not immunogenic, are easily synthesized, can be chemically modified, and are not very demanding in storage conditions. It is possible to select an aptamer that specifically binds to any target cell, oncolytic virus, or molecule using the SELEX technology. This review comprehensively highlights the most important research and methodological approaches related to oncolytic viruses and nucleic acid aptamers. Here, we also analyze possible future research directions for combining these two methodologies to improve the effectiveness of cancer virotherapy.

Homocystamide Conjugates of Human Serum Albumin as a Platform to Prepare Bimodal Multidrug Delivery Systems for Boron Neutron Capture Therapy.

Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro-closo-dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 µM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro-closo-dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.

Molecular Mechanisms of Drug Resistance in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, is highly resistant to conventional radiation and chemotherapy, and is not amenable to effective surgical resection. The present review summarizes recent advances in our understanding of the molecular mechanisms of therapeutic resistance of GBM to already known drugs, the molecular characteristics of glioblastoma cells, and the barriers in the brain that underlie drug resistance. We also discuss the progress that has been made in the development of new targeted drugs for glioblastoma, as well as advances in drug delivery across the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB).

Tumor Cell-Specific 2'-Fluoro RNA Aptamer Conjugated with Closo-Dodecaborate as A Potential Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a binary radiotherapeutic approach to the treatment of malignant tumors, especially glioblastoma, the most frequent and incurable brain tumor. For successful BNCT, a boron-containing therapeutic agent should provide selective and effective accumulation of 10B isotope inside target cells, which are then destroyed after neutron irradiation. Nucleic acid aptamers look like very prospective candidates for carrying 10B to the tumor cells. This study represents the first example of using 2'-F-RNA aptamer GL44 specific to the human glioblastoma U-87 MG cells as a boron delivery agent for BNCT. The closo-dodecaborate residue was attached to the 5'-end of the aptamer, which was also labeled by the fluorophore at the 3'-end. The resulting bifunctional conjugate showed effective and specific internalization into U-87 MG cells and low toxicity. After incubation with the conjugate, the cells were irradiated by epithermal neutrons on the Budker Institute of Nuclear Physics neutron source. Evaluation of the cell proliferation by real-time cell monitoring and the clonogenic test revealed that boron-loaded aptamer decreased specifically the viability of U-87 MG cells to the extent comparable to that of 10B-boronophenylalanine taken as a control. Therefore, we have demonstrated a proof of principle of employing aptamers for targeted delivery of boron-10 isotope in BNCT. Considering their specificity, ease of synthesis, and large toolkit of chemical approaches for high boron-loading, aptamers provide a promising basis for engineering novel BNCT agents.

Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells.

BACKGROUND: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. METHODS: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. RESULTS: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). CONCLUSIONS: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

Molecular characterization of Mycoplasma pneumoniae infections in Moscow from 2015 to 2018.

The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.

Characterization of extensively drug-resistant Mycobacterium tuberculosis isolates circulating in Siberia.

BACKGROUND: The spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis compromises effective control of tuberculosis (TB) in Siberia. Early identification of drug-resistant isolates is, therefore, crucial for effective treatment of this disease. The aim of this study was to conduct drug susceptibility testing and identify mutations in drug resistance genes in clinical isolates of M. tuberculosis from some TB patients presenting for treatment in Siberia. METHODS: Thirty randomly selected clinical isolates of M. tuberculosis were obtained from the Novosibirsk Research Institute of Tuberculosis, Russia. Isolates were screened for drug resistance and characterized by variable number of tandem repeats (VNTR)-typing using 15 standard and four additional loci. Deligotyping on multiple large sequences was performed using 10 loci. RESULTS: Twenty-nine of the isolates were assigned XDR status. Twenty-eight isolates belonged to the M. tuberculosis Beijing family, from which 11 isolates were considered the M11 type (39%), two the M2 type (7%), and one the M33 type (3%). Seventeen isolates (60.7%) from this family exhibited unique genetic patterns. The remaining two isolates belonged to the Latino-American Mediterranean family. Gene sequences (rpoB, katG, rrs, rpsL, tlyA, gidB, gyrA, gyrB) were analyzed to identify mutations that confer resistance to rifampicin, isoniazid, amikacin, kanamycin, capreomycin, and ofloxacin. The most common mutations among the XDR isolates were S531L in RpoB, S315T in KatG, various codon 94 mutations in gyrA, A90V in GyrA, K43R in RpsL, and 1401 A → G in rrs; these confer resistance to rifampicin, isoniazid, ofloxacin, streptomycin and kanamycin/capreomycin, respectively. There was high congruence between the two typing methods (VNTR typing and deligotyping) and RD105, RD149, RD152, RD181, and RD207 regions of difference were absent from the 28 Beijing family isolates. CONCLUSIONS: Deligotyping can be used for rapid and reliable screening of M. tuberculosis isolates, followed by more in-depth genotyping. Identification of Beijing family isolates with extensive drug resistance confirms that such strains have epidemiological importance in Siberia. Rapid detection of mutations that lead to drug resistance should facilitate selection of effective drug therapies, and the development of early prevention strategies to combat this infection.

Genetic variation of Mycobacterium tuberculosis circulating in Kharkiv Oblast, Ukraine.

BACKGROUND: A persistent increase of tuberculosis cases has recently been noted in the Ukraine. The reported incidence of drug-resistant isolates of M. tuberculosis is growing steadily; however, data on the genetic variation of isolates of M. tuberculosis circulating in northern Ukraine and on the spectrum and frequency of occurrence of mutations determining resistance to the principal anti-tuberculosis drugs isoniazid and rifampicin have not yet been reported. METHODS: Isolates of M. tuberculosis from 98 tuberculosis patients living in Kharkiv Oblast (Ukraine) were analyzed using VNTR- and RFLP-IS6110-typing methods. Mutations associated with resistance to rifampicin and isoniazid were detected by RFLP-PCR methods, and also confirmed by sequencing. RESULTS: We identified 75 different genetic profiles. Thirty four (34%) isolates belonged to the Beijing genotype and 23 (23%) isolates belonged to the LAM family. A cluster of isolates belonging to the LAM family had significant genetic heterogeneity, indicating that this family had an ancient distribution and circulation in this geographical region. Moreover, we found a significant percentage of the isolates (36%) belonged to as yet unidentified families of M. tuberculosis or had individual non-clustering genotypes. Mutations conferring rifampicin and isoniazid resistance were detected in 49% and 54% isolates, respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene were predominant among drug-resistant isolates. An association was found for belonging to the LAM strain family and having multiple drug resistance (R = 0.27, p = 0.0059) and also for the presence of a mutation in codon 531 of the rpoB gene and belonging to the Beijing strain family (R = 0.2, p = 0.04). CONCLUSIONS: Transmission of drug-resistant isolates seems to contribute to the spread of resistant TB in this oblast. The Beijing genotype and LAM genotype should be seen as a major cause of drug resistant TB in this region.