The Recombinant Oncolytic Virus VV-GMCSF-Lact and Chemotherapy Drugs against Human Glioma.

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

Combination of Oncolytic Virotherapy with Different Antitumor Approaches against Glioblastoma.

Glioblastoma is one of the most malignant and aggressive tumors of the central nervous system. Despite the standard therapy consisting of maximal surgical resection and chemo- and radiotherapy, the median survival of patients with this diagnosis is about 15 months. Oncolytic virus therapy is one of the promising areas for the treatment of malignant neoplasms. In this review, we have focused on emphasizing recent achievements in virotherapy, both as a monotherapy and in combination with other therapeutic schemes to improve survival rate and quality of life among patients with glioblastoma.

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.

Human Blood Extracellular Vesicles Activate Transcription of NF-kB-Dependent Genes in A549 Lung Adenocarcinoma Cells.

Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs' RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases.

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 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.

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.

Modification of a Tumor-Targeting Bacteriophage for Potential Diagnostic Applications.

BACKGROUND: Tumor-targeting bacteriophages can be used as a versatile new platform for the delivery of diagnostic imaging agents and therapeutic cargo. This became possible due to the development of viral capsid modification method. Earlier in our laboratory and using phage display technology, phages to malignant breast cancer cells MDA-MB 231 were obtained. The goal of this study was the optimization of phage modification and the assessment of the effect of the latter on the efficiency of phage particle penetration into MDA-MB 231 cells. METHODS: In this work, we used several methods, such as chemical phage modification using FAM-NHS ester, spectrophotometry, phage amplification, sequencing, phage titration, flow cytometry, and confocal microscopy. RESULTS: We performed chemical phage modification using different concentrations of FAM-NHS dye (0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM). It was shown that with an increase of the modification degree, the phage titer decreases. The maximum modification coefficient of the phage envelope with the FAM-NHS dye was observed with 4 mM modifying agent and had approximately 804,2 FAM molecules per phage. Through the immunofluorescence staining and flow cytometry methods, it was shown that the modified bacteriophage retains the ability to internalize into MDA-MB-231 cells. The estimation of the number of phages that could have penetrated into one tumor cell was conducted. CONCLUSIONS: Optimizing the conditions for phage modification can be an effective strategy for producing tumor-targeting diagnostic and therapeutic agents, i.e., theranostic drugs.

Double Recombinant Vaccinia Virus: A Candidate Drug against Human Glioblastoma.

Glioblastoma is one of the most aggressive brain tumors. Given the poor prognosis of this disease, novel methods for glioblastoma treatment are needed. Virotherapy is one of the most actively developed approaches for cancer therapy today. VV-GMCSF-Lact is a recombinant vaccinia virus with deletions of the viral thymidine kinase and growth factor genes and insertions of the granulocyte-macrophage colony-stimulating factor and oncotoxic protein lactaptin genes. The virus has high cytotoxic activity against human cancer cells of various histogenesis and antitumor efficacy against breast cancer. In this work, we show VV-GMCSF-Lact to be a promising therapeutic agent for glioblastoma treatment. VV-GMCSF-Lact effectively decreases the viability of glioblastoma cells of both immortalized and patient-derived cultures in vitro, crosses the blood-brain barrier, selectively replicates into orthotopically transplanted human glioblastoma when intravenously injected, and inhibits glioblastoma xenograft and metastasis growth when injected intratumorally.

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.

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).

Method of Measuring High-LET Particles Dose.

In boron neutron capture therapy, the total absorbed dose is the sum of four dose components with different relative biological effectiveness (RBE): boron dose, "nitrogen" dose, fast neutron dose and γ-ray dose. We present a new approach for measuring the first three doses. In this work, we provide the details of this method of dose measurement and results when this proposed method is employed.

Boron neutron capture therapy: Current status and future perspectives.

The development of new accelerators has given a new impetus to the development of new drugs and treatment technologies using boron neutron capture therapy (BNCT). We analyzed the current status and future directions of BNCT for cancer treatment, as well as the main issues related to its introduction. This review highlights the principles of BNCT and the key milestones in its development: new boron delivery drugs and different types of charged particle accelerators are described; several important aspects of BNCT implementation are discussed. BCNT could be used alone or in combination with chemotherapy and radiotherapy, and it is evaluated in light of the outlined issues. For the speedy implementation of BCNT in medical practice, it is necessary to develop more selective boron delivery agents and to generate an epithermal neutron beam with definite characteristics. Pharmacological companies and research laboratories should have access to accelerators for large-scale screening of new, more specific boron delivery agents.

Interplay Between Mitophagy and Apoptosis Defines a Cell Fate Upon Co-treatment of Breast Cancer Cells With a Recombinant Fragment of Human κ-Casein and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

A recombinant fragment of human κ-Casein, termed RL2, induces cell death of breast cancer cells; however, molecular mechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-term co-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis.

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.

Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties.

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2's activity remain unclear. To elucidate the structure-activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.

Cytotoxic and Antitumor Activity of Lactaptin in Combination with Autophagy Inducers and Inhibitors.

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Nucleotide Modifications Decrease Innate Immune Response Induced by Synthetic Analogs of snRNAs and snoRNAs.

Short nuclear regulatory RNAs play a key role in the main stages of maturation of the precursors of the major RNA species. Small nuclear RNAs (snRNAs) form the core of the spliceosome and are responsible for the splicing of pre-mRNA molecules. Small nucleolar RNAs (snoRNAs) direct post-transcriptional modification of pre-rRNAs. A promising strategy for the development of non-coding RNA (ncRNAs) mimicking molecules is the introduction of modified nucleotides, which are normally present in natural ncRNAs, into the structure of synthetic RNAs. We have created a set of snoRNAs and snRNA analogs and studied the effect of base modifications, specifically, pseudouridine (Ψ) and 5-methylcytidine (m5C), on the immune-stimulating and cytotoxic properties of these RNAs. Here, we performed a whole-transcriptome study of the influence of synthetic snoRNA analogs with various modifications on gene expression in human cells. Moreover, we confirmed the role of PKR in the recognition of snoRNA and snRNA analogs using the short hairpin RNA (shRNA) technique. We believe that the data obtained will contribute to the understanding of the role of nucleotide modification in ncRNA functions, and can be useful for creating the agents for gene regulation based on the structure of natural snoRNAs and snRNAs.