Recent Advances on RNA-Based Molecular Therapies for Tumour Cell Targeting.

RNA-based gene therapy has been widely used for various diseases, and extensive studies have proved that suitable delivery routes greatly help the development of RNA therapeutics. Identifying a safe and effective delivery system is key to realizing RNA therapeutics' clinical translation. Inhalation is a non-invasive pulmonary delivery modality that can enhance the retention of therapeutic agents in the lungs with negligible toxicity, thereby improving patient compliance. Inhaled RNA therapeutics are increasingly becoming an area of focus for researchers; however, only several clinical trials have explored inhaled delivery of RNA for pulmonary diseases. This review presents an overview of recent advances in inhaled delivery systems for RNA therapeutics, including viral and nonviral systems, highlighting state of the art regarding inhalation in the messenger RNA (mRNA) field. We also summarize the applications of mRNA inhalants in infectious and other lung diseases. Simultaneously, the research progresses on small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and different types of RNA are also discussed to provide new strategies for developing RNA inhalation therapy. Finally, we clarify the challenges inhaled RNA-based therapeutics face before their widespread adoption and provide insights to help advance this exciting field to the bedside.

Review: RNA-based diagnostic markers discovery and therapeutic targets development in cancer

RNA-Based Therapeutics: Ready for Delivery?

The role of RNA in the treatment of the diabetic foot has received increasing attention as RNA therapy has been used in a variety of ways. This article explores various delivery methods for different types of RNA, with a focus on their mechanisms, efficiency, and potential applications. Discussed encapsulation, adsorption, layer by layer assembly, covalent binding, and targeting strategies involving MiRNA, SiRNA, mRNA, and LncRNA. Analyze the advantages and disadvantages of each method in targeted delivery and release of RNA, and point out their potential in clinical applications for diabetic wound.

Advanced technological tools to study multidrug resistance in cancer.

Emerging Role of Semaphorins as Major Regulatory Signals and Potential Therapeutic Targets in Cancer

ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR.

RNA methylation is widespread in nature. Abnormal expression of proteins associated with RNA methylation is strongly associated with a number of human diseases including cancer. Increasing evidence suggests that targeting RNA methylation holds promise for cancer treatment. This review specifically describes several common RNA modifications, such as the relatively well‐studied N6‐methyladenosine, as well as 5‐methylcytosine and pseudouridine (Ψ). The regulatory factors involved in these modifications and their roles in RNA are also comprehensively discussed. We summarise the diverse regulatory functions of these modifications across different types of RNAs. Furthermore, we elucidate the structural characteristics of these modifications along with the development of specific inhibitors targeting them. Additionally, recent advancements in small molecule inhibitors targeting RNA modifications are presented to underscore their immense potential and clinical significance in enhancing therapeutic efficacy against cancer.Key PointsIn this paper, several important types of RNA modifications and their related regulatory factors are systematically summarised.Several regulatory factors related to RNA modification types were associated with cancer progression, and their relationships with cancer cell migration, invasion, drug resistance and immune environment were summarised.In this paper, the inhibitors targeting different regulators that have been proposed in recent studies are summarised in detail, which is of great significance for the development of RNA modification regulators and cancer treatment in the future.

Approaches to develop effective drugs to kill cancer cells are mainly focused either on the improvement of the currently used chemotherapeutics or on the development of targeted therapies aimed at the selective destruction of cancer cells by steering specific molecules and/or enhancing the immune response. The former strategy is limited by its genotoxicity and severe side effects, while the second one is not always effective due to tumor cell heterogeneity and variability of targets in cancer cells. Between these two strategies, several approaches target different types of RNA in tumor cells. RNA degradation alters gene expression at different levels inducing cell death. However, unlike DNA targeting, it is a pleotropic but a non-genotoxic process. Among the ways to destroy RNA, we find the use of ribonucleases with antitumor properties. In the last few years, there has been a significant progress in the understanding of the mechanism by which these enzymes kill cancer cells and in the development of more effective variants. All the approaches seek to maintain the requirements of the ribonucleases to be specifically cytotoxic for tumor cells. These requirements start with the competence of the enzymes to interact with the cell membrane, a process that is critical for their internalization and selectivity for tumor cells and continue with the downstream effects mainly relying on changes in the RNA molecular profile, which are not only due to the ribonucleolytic activity of these enzymes. Although the great improvements achieved in the antitumor activity by designing new ribonuclease variants, some drawbacks still need to be addressed. In the present review, we will focus on the known mechanisms used by ribonucleases to kill cancer cells and on recent strategies to solve the shortcomings that they show as antitumor agents, mainly their pharmacokinetics.

An increasing body of evidence supports the notion that long noncoding RNAs (lncRNAs) play important roles in diseases including cancer. MALAT1 (also called NEAT2) was originally identified in tumors of highly metastatic non-small cell lung cancer (NSCLC), however, the functional role of MALAT1 in carcinogenesis has remained largely unknown. However, MALAT1 is highly expressed in various types of cancer and is implicated in the regulation of alternative splicing. To determine if MALAT1 constitutes an important driver of tumor growth in vivo, we developed potent antisense oligonucleotides (ASOs) that were able to achieve strong inhibition of MALAT1 in the tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses leading to >95% inhibition of MALAT1 RNA in the liver. Inhibition of MALAT1 expression in tumor and tumor-associated stromal cells was determined by both q-RT-PCR using species-specific probe/primer sets and/or in situ ‘viewRNA’ technology, where target knockdown can be visualized on a cell by cell basis. Systemic administration of mouse MALAT1 ASOs resulted in a decrease in the numbers of polyps and proliferation index (BrdU (+)) in the small intestine of Apcmin mouse model of colon cancer and correlated well with MALAT1 inhibition in the polyps. Mouse MALAT1 ASOs were also effective in a DEN-induced HCC model, where ASO treatment reduced the target RNA ~ 90% in tumor cells with a concomitant decrease in tumor numbers, while control ASO had no effects on either measure. In addition, higher expression of MALAT1 in non-treated tumors compared to adjacent normal hepatocytes was also clearly visualized by the in situ ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size in TRAMP mouse model of prostate cancer, where the target RNA was decreased by 80% in tumor cells. In a human NSCLC patient-derived xenograft model, both significant MALAT1 RNA reduction and a delay in tumor growth were achieved after MALAT1 ASO treatment. The effects of MALAT1 downregulation by ASO were not limited to the inhibition of tumor growth alone. MALAT1 ASO treatment not only inhibited the growth of primary tumors in the EBC-1 human NSCLC xenograft model, but also resulted in a decrease in lung metastasis as measured by micro CT scanning. Furthermore, cross-species MALAT1 ASOs greatly improved the survival of animals bearing Hep3B human hepatocellular carcinoma (HCC) tumor orthotopically (48.5 days with control ASO vs 88 days with MALAT1 ASO, p=0.005). Taken together, these results demonstrate previously undiscovered roles of MALAT1 as an important regulator in vivo tumor growth and metastasis and suggest that selective inhibition of MALAT1 by ASO could have therapeutic value for the cancer treatment. Citation Format: Jeff Hsu, Guobin He, Gourab Bhattacharjee, Tianyuan Zhou, Chris May, Xiaokun Xiao, Gene Hung, Brett P. Monia, A. Robert MacLeod, Youngsoo Kim. Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B23.

RNA drugs and nonviral nanocarriers in oncology - present status and emerging concepts.

Information processing tools and bioinformatics software have markedly advanced the ability of researchers to process and analyze biological data. Data from the genomes of humans and model organisms aid researchers to identify topics to study, which in turn improves predictive accuracy, facilitates the identification of relevant genes and simplifies the validation of laboratory data. The objective of the present study was to investigate the regulatory network constituted by long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNA in hepatocellular carcinoma (HCC). Microarray data from HCC datasets were downloaded from The Cancer Genome Atlas database, and the Limma package in R was used to identify the differentially expressed genes (DEGs) between HCC and normal samples. Gene ontology enrichment analysis of DEGs was conducted using the Database for Annotation, Visualization, and Integrated Discovery. TargetScan, microcosm, miRanda, miRDB and PicTar were used to predict target genes. lncRNAs associated with HCC were probed using the lncRNASNP database, and a lncRNA-miRNA-mRNA regulatory network was visualized using Cytoscape. The present study identified 114 differentially expressed miRNAs and 2,239 differentially expressed mRNAs; of these, 725 were downregulated genes that were primarily involved in complement and coagulation cascades, fatty acid metabolism and butanoate metabolism, among others. The remaining 1,514 were upregulated genes principally involved in DNA replication, oocyte meiosis and homologous recombination, among others. Through the integrated analysis of associations between different types of RNAs and target gene prediction, the present study identified 203 miRNA-mRNA pairs, including 28 miRNAs and 170 mRNAs, and identified 348 lncRNA-miRNA pairs, containing 28 miRNAs. Therefore, owing to the association between lncRNAs-miRNAs-mRNAs, the present study screened out 2,721 regulatory associations. The data in the present study provide a comprehensive bioinformatic analysis of genes, functions and pathways that may be involved in the pathogenesis of HCC.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9), an adaptive microbial immune system, has been exploited as a robust, accurate, efficient and programmable method for genome targeting and editing. This innovative and revolutionary technique can play a significant role in animal modeling, in vivo genome therapy, engineered cell therapy, cancer diagnosis and treatment. The CRISPR/Cas9 endonuclease system targets a specific genomic locus by single guide RNA (sgRNA), forming a heteroduplex with target DNA. The Streptococcus pyogenes Cas9/sgRNA:DNA complex reveals a bilobed architecture with target recognition and nuclease lobes. CRISPR/Cas9 assembly can be hijacked, and its nanoformulation can be engineered as a delivery system for different clinical utilizations. However, the efficient and safe delivery of the CRISPR/Cas9 system to target tissues and cancer cells is very challenging, limiting its clinical utilization. Viral delivery strategies of this system may have many advantages, but disadvantages such as immune system stimulation, tumor promotion risk and small insertion size outweigh these advantages. Thus, there is a desperate need to develop an efficient non-viral physical delivery system based on simple nanoformulations. The delivery strategies of CRISPR/Cas9 by a nanoparticle-based system have shown tremendous potential, such as easy and large-scale production, combination therapy, large insertion size and efficient in vivo applications. This review aims to provide in-depth updates on Streptococcus pyogenic CRISPR/Cas9 structure and its mechanistic understanding. In addition, the advances in its nanoformulation-based delivery systems, including lipid-based, polymeric structures and rigid NPs coupled to special ligands such as aptamers, TAT peptides and cell-penetrating peptides, are discussed. Furthermore, the clinical applications in different cancers, clinical trials and future prospects of CRISPR/Cas9 delivery and genome targeting are also discussed.

Exosomes have been widely applied to the delivery of RNA and small molecules currently. However, the low targeting and specificity greatly limited the effect of exosome delivery. Here we designed an exosome that can perform the targeted delivery of two different types of RNA. Based on the mesenchymal stem cells (MSCs) derived exosomes, the RNA delivery system of targeted dendritic cells (DC-Exosome) was constructed, using the layer by layer self-assembly. DC-Exosomes can specifically bind to DCs, while guiding the endocytosis of chimeras and exosome. Then aptamer/siRNA chimera was cut into mTOR siRNA by Dicer, and microRNA was released from exosome under lysosomal digestion. SIGN aptamer performed the rapid induction of immune tolerance, and later mTOR siRNA was formed to inhibit mTOR pathway and suppress immune responses. Exosomes could maintain long time-stability after PEG-PEI polyplexes modification and promote HLA-G expression in DCs continuously. Animal experiments showed that DC-Exosomes could induce immune tolerance at 3, 7, and 14 days after skin transplantation. Compared with the microRNA-Exosome group, the number of CD11c+ DCs in DC-Exosome group decreased, while the proportion of HLA-G+ DCs increased remarkably. In conclusion, we constructed a new exosome-based targeted delivery system which could effectively induce the immune tolerance in transplantation.

Integrin receptors have remained as a key subject of interest in the pharmaceutical industry for the last few years. There are a total of 24 different types of integrin heterodimers. Each of these heterodimers plays important role in various biological processes that are inherent to different pathological conditions. As a result, integrin receptors have been extensively evaluated for their role in therapeutic targeting. There are different classes of inhibitors against integrin receptors and this review provides an overview on different classes of integrin inhibitors that are currently available. A number of review articles have been written on the possible application of integrin receptors in therapeutic targeting. Many of these articles have heavily emphasized on the importance of αvβ3 & αvβ5 receptors as major pharmaceutical target in cancer but little emphasis has been given on the importance of other integrin receptors, such as α5β1, αIIbβ3, α4β7, αvβ6 etc. While this review gives due importance to both αvβ3 & αvβ5 receptors and provides an historical perspective on how these two receptors have evolved as a potential target for cancer, significant emphasis has also been given on the other integrin receptors that have started enjoying the status of important drug target over the course of last few years. Effort has been maintained to discuss briefly on the key physiological basis of their importance as drug target. For example, involvement of αvβ3 in angiogenesis has made it a therapeutic target for the treatment of cancer. At the same time expression of this receptor on the surface of osteoclast has made it a target for the treatment of osteoporosis. Thus, emphasis has been given on discussing the role of the integrin receptors in different disease conditions followed by specific examples of drug molecules that have been trialed against these receptors. While hundreds of candidate molecules have been developed against different integrin receptors only a handful of them has been subject to phase-III clinical trial. That necessitates careful consideration of certain concerns that are associated with direct targeting of integrins and thus has also been an important goal of this review. In the last few years application of integrin receptors have extended beyond mere therapeutic targeting. Several integrin receptors are currently are studied for their potential of aiding at diagnostic imaging and drug delivery. In this review a brief overview has also been provided on how integrin are being targeted for diagnostic imaging and drug delivery with relevant examples. Thus the primary aim of this review has been to provide an comprehensive overview on the broad scope of application that integrin receptors have in the field of medical science.