RNAi-based therapeutics and tumor targeted delivery in cancer.

Over the past decade, non-coding RNA-based therapeutics have proven as a great potential for the development of targeted therapies for cancer and other diseases. The discovery of the critical function of microRNAs (miRNAs) has generated great excitement in developing miRNA-based therapies. The dysregulation of miRNAs contributes to the pathogenesis of various human diseases and cancers by modulating genes that are involved in critical cellular processes, including cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, drug resistance, and tumorigenesis. miRNA (miRNA mimic, anti-miRNA/antagomir) and small interfering RNA (siRNA) can inhibit the expression of any cancer-related genes/mRNAs with high specificity through RNA interference (RNAi), thus representing a remarkable therapeutic tool for targeted therapies and precision medicine. siRNA and miRNA-based therapies have entered clinical trials and recently three novel siRNA-based therapeutics were approved by the Food and Drug Administration (FDA), indicating the beginning of a new era of targeted therapeutics. The successful clinical applications of miRNA and siRNA therapeutics rely on safe and effective nanodelivery strategies for targeting tumor cells or tumor microenvironment. For this purpose, promising nanodelivery/nanoparticle-based approaches have been developed using a variety of molecules for systemic administration and improved tumor targeted delivery with reduced side effects. In this review, we present an overview of RNAi-based therapeutics, the major pharmaceutical challenges, and the perspectives for the development of promising delivery systems for clinical translation. We also highlight the passive and active tumor targeting nanodelivery strategies and primarily focus on the current applications of nanoparticle-based delivery formulations for tumor targeted RNAi molecules and their recent advances in clinical trials in human cancers.

Non-coding RNAs: Emerging from the discovery to therapeutic applications.

The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.

Single bioengineered ncRNA molecule for dual-targeting toward the control of non-small cell lung cancer patient-derived xenograft tumor growth.

Lung cancer remains the leading cause of cancer deaths worldwide and accounts for more than 22% of all cancer-related deaths in the US. Developing new therapies is essential to combat against deadly lung cancer, especially the most common type, non-small cell lung cancer (NSCLC). With the discovery of genome-derived functional small noncoding RNA (ncRNA), namely microRNAs (miRNA or miR), restoration of oncolytic miRNAs lost or downregulated in NSCLC cells represents a new therapeutic strategy. Very recently, we have developed a novel technology that achieves in vivo fermentation production of bioengineered miRNA agents (BERA) for research and development. In this study, we aimed at simultaneously introducing two miRNAs into NSCLC cells by using single recombinant "combinatorial BERA" (CO-BERA) molecule. Our studies show that single CO-BERA molecule (e.g., let-7c/miR-124) was successfully processed to two miRNAs (e.g., let-7c-5p and miR-124-3p) to combinatorially regulate the expression of multiple targets (e.g., RAS, VAMP3 and CDK6) in human NSCLC cells, exhibiting greater efficacy than respective BERA miRNAs in the inhibition of cell viability and colony formation. Furthermore, we demonstrate that CO-BERA let-7c/miR-124-loaded lipopolyplex nanomedicine was the most effective among tested RNAs in the control of tumor growth in NSCLC patient-derived xenograft mouse models. The anti-tumor activity of CO-BERA let-7c/miR-124 was associated with the suppression of RAS and CDK6 expression, and enhancement of apoptosis. These results support the concept to use single ncRNA agent for dual-targeting and offer insight into developing new RNA therapeutics for the treatment of lethal NSCLC.

Improving the therapeutic efficiency of noncoding RNAs in cancers using targeted drug delivery systems.

The delivery of noncoding (nc)RNA to target cancer stem cells and metastatic tumors has shown many positive outcomes, resulting in improved and more efficient treatment strategies. The success of therapeutic RNA depends solely on passing cellular barriers to reach the target site, where it binds to the mRNA of the interest. By 2018, 20 clinical trials had been initiated, most focusing on cancer and diabetes, with some progressing to Phase II clinical trials testing the safety and efficacy of small interfering (si)RNA. Many challenges limit RNA interference (RNAi) and miRNA usage in vivo; therefore, various approaches have been developed to promote ncRNA efficiency and stability. In this review, we focus on targeting the tumor microenvironment (TME) via the modification of delivery systems utilizing nanotechnology-based delivery approaches.

Noncoding RNAs in cancer therapy resistance and targeted drug development.

Noncoding RNAs (ncRNAs) represent a large segment of the human transcriptome and have been shown to play important roles in cellular physiology and disease pathogenesis. Increasing evidence on the functional roles of ncRNAs in cancer progression emphasizes the potential of ncRNAs for cancer treatment. Here, we summarize the roles of ncRNAs in disease relapse and resistance to current standard chemotherapy and radiotherapy; the current research progress on ncRNAs for clinical and/or potential translational applications, including the identification of ncRNAs as therapeutic targets; therapeutic approaches for ncRNA targeting; and ncRNA delivery strategies in potential clinical translation. Several ongoing clinical trials of novel RNA-based therapeutics were also emphasized. Finally, we discussed the perspectives and obstacles to different target combinations, delivery strategies, and system designs for ncRNA application. The next approved nucleic acid drug to treat cancer patients may realistically be on the horizon.

Rodlike Supramolecular Nanoassemblies of Degradable Poly(Aspartic Acid) Derivatives and Hydroxyl-Rich Polycations for Effective Delivery of Versatile Tumor-Suppressive ncRNAs.

The delivery of tumor-suppressive noncoding RNAs (ncRNAs) including short ncRNAs (i.e., miRNAs) and long ncRNAs (lncRNAs) is put forward to treat tumors. In this work, novel rodlike supramolecular nanoassemblies (CNC @CB[8] @ PGEA) of degradable poly(aspartic acid) (PAsp) derivatives-grafted cellulose nanocrystals (CNCs) and hydroxyl-rich polycations (ethanolamine-functionalized poly(glycidyl methacrylate), PGEA) are proposed via typical cucurbit[8]uril (CB[8])-based host-guest interactions for delivery of different ncRNAs to treat hepatocellular carcinoma (HCC). Spindly CNCs, one kind of natural polysaccharide nanoparticles, possess good biocompatibility and unique physico-chemical properties. PGEA with abundant hydroxyl groups is one promising gene carrier with low cytotoxicity. PAsp can benefit the disassembly and degradability of nanoassemblies within cells. CNC @ CB[8]@PGEA combines the different unique properties of CNC, PGEA, and PAsp. CNC @ CB[8] @ PGEA effectively complexes the expression constructs of miR-101 (plasmid pc3.0-miR-101) and lncRNA MEG3 (plasmid pc3.0-MEG3). CNC @ CB[8] @ PGEA produces much better transfection performances than PGEA-containing assembly units. In addition, the codelivery system of CNC @ CB[8] @ PGEA/(pc3.0-MEG3+pc3.0-miR-101) nanocomplexes demonstrates better efficacy in suppressing HCC than CNC @ CB[8] @ PGEA/pc3.0-MEG3 or CNC @ CB[8] @ PGEA/pc3.0-miR-101 nanocomplexes alone. Such rodlike supramolecular nanoassemblies will provide a promising means to produce efficient delivery vectors of versatile tumor-suppressive nucleic acids.

Targeting Promoter-Associated Noncoding RNA In Vivo.

There are many classes of noncoding RNAs (ncRNAs), with wide-ranging functionalities (e.g., RNA editing, mediation of mRNA splicing, ribosomal function). MicroRNAs (miRNAs) and long ncRNAs (lncRNAs) are implicated in a wide variety of cellular processes, including the regulation of gene expression. Incorrect expression or mutation of lncRNAs has been reported to be associated with several disease conditions, such a malignant transformation in humans. Importantly, pivotal players in tumorigenesis and cancer progression, such as c-Myc, may be regulated by lncRNA at promoter level. The function of lncRNA can be reduced with antisense oligonucleotides that sequester or degrade mature lncRNAs. In alternative, lncRNA transcription can be blocked by small interference RNA (RNAi), which had acquired, recently, broad interested in clinical applications. In vivo-jetPEI™ is a linear polyethylenimine mediating nucleic acid (DNA, shRNA, siRNA, oligonucelotides) delivery with high efficiency. Different in vivo delivery routes have been validated: intravenous (IV), intraperitoneal (IP), intratumoral, subcutaneous, topical, and intrathecal. High levels of nucleic acid delivery are achieved into a broad range of tissues, such as lung, salivary glands, heart, spleen, liver, and prostate upon systemic administration. In addition, in vivo-jetPEI™ is also an efficient carrier for local gene and siRNA delivery such as intratumoral or topical application on the skin. After systemic injection, siRNA can be detected and the levels can be validated in target tissues by qRT-PCR. Targeting promoter-associated lncRNAs with siRNAs (small interfering RNAs) in vivo is becoming an exciting breakthrough for the treatment of human disease.

Non-coding RNAs: Therapeutic Strategies and Delivery Systems.

The vast majority of the human genome is transcribed into RNA molecules that do not code for proteins, which could be small ones approximately 20 nucleotide in length, known as microRNAs, or transcripts longer than 200 bp, defined as long noncoding RNAs. The prevalent deregulation of microRNAs in human cancers prompted immediate interest on the therapeutic value of microRNAs as drugs and drug targets. Many features of microRNAs such as well-defined mechanisms, and straightforward oligonucleotide design further make them attractive candidates for therapeutic development. The intensive efforts of exploring microRNA therapeutics are reflected by the large body of preclinical studies using oligonucleotide-based mimicking and blocking, culminated by the recent entry of microRNA therapeutics in clinical trial for several human diseases including cancer. Meanwhile, microRNA therapeutics faces the challenge of effective and safe delivery of nucleic acid therapeutics into the target site. Various chemical modifications of nucleic acids and delivery systems have been developed to increase targeting specificity and efficacy, and reduce the associated side effects including activation of immune response. Recently, long noncoding RNAs become attractive targets for therapeutic intervention because of their association with complex and delicate phenotypes, and their unconventional pharmaceutical activities such as capacity of increasing output of proteins. Here I discuss the general therapeutic strategies targeting noncoding RNAs, review delivery systems developed to maximize noncoding RNA therapeutic efficacy, and offer perspectives on the future development of noncoding RNA targeting agents for colorectal cancer.

Emerging potential of exosomes and noncoding microRNAs for the treatment of neurological injury/diseases.

Recent discoveries of cellular generation of exosomes, small (∼ 30 - 100 nm) complex lipid membrane structures which encapsulate and transport proteins, RNAs, including microRNAs (miRNAs) have provided new insight in how cells within organisms communicate. These discoveries will likely have a major impact on the treatment of disease, with cancers and neurological diseases as evident targets. Exosomes provide a major medium of intercellular communications and thereby, there being a potential by altering communications and instructions for protein production, we can employ exosomes to treat diseases. We now have an opportunity to treat neurological disease by modifying intercellular communication networks. Recent work demonstrating that the therapeutic benefit provided by stem cells for the treatments of stroke and traumatic brain injury depend on their generation and release of exosomes provides a foundation for exosome-based therapy. Cell-free exosomes have also been recently employed to effectively treat stroke and brain trauma. The content of exosomes, particularly their miRNA cargo which can concurrently impact the post-transcriptional regulation of many genes, can be regulated. We are at the cusp of capitalizing on this important means of intercellular communications for the treatment of diseases, such as cancers and neurological diseases, among many others.

The prrF-encoded small regulatory RNAs are required for iron homeostasis and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence.

A novel neuroprotective therapy for Parkinson's disease using a viral noncoding RNA that protects mitochondrial complex I activity.

Parkinson's disease (PD) is a neurodegenerative disorder that results in the loss of nigrostriatal dopamine neurons. The etiology of this cell loss is unknown, but it involves abnormalities in mitochondrial function. In this study, we have demonstrated that the administration of a novel noncoding p137 RNA, derived from the human cytomegaloviral ß2.7 transcript, can prevent and rescue dopaminergic cell death in vitro and in animal models of PD by protecting mitochondrial Complex I activity. Furthermore, as this p137 RNA is fused to a rabies virus glycoprotein peptide that facilitates delivery of RNA across the blood-brain barrier, such protection can be achieved through a peripheral intravenous administration of this agent after the initiation of a dopaminergic lesion. This approach has major implications for the potential treatment of PD, especially given that this novel agent could have the same protective effect on all diseased neurons affected as part of this disease process, not just the dopaminergic nigrostriatal pathway.

MicroRNAs: a new paradigm on molecular urological oncology.

MicroRNAs (miRNA) are a class of naturally occurring, small, noncoding RNAs that function as specific repressors of protein-encoding genes, being pivotal regulators of development and cellular homeostasis. They have been functionally classified as proto-oncogenes or tumor suppressors and are aberrantly expressed in different cancer types. Deregulation of these so-called "cancerous" miRNAs can figure prominently in tumor initiation and progression. The Human miRNA profile is under development, and it is of concern that there is so little agreement on the miRNAs found in each of the studies. Bioinformatics progress and efficient delivery are essential to overcome barriers to miRNA use in clinical practice.

Biodegradable polymer-mediated sh/siRNA delivery for cancer studies.

Discovery of RNA interference (RNAi)-mediated specific gene silencing has raised hope for cancer therapy. Unfortunately, the execution of RNAi by delivering small-interfering RNA (siRNA) or small hairpin RNA (shRNA) remains a prime challenge. A methodical evaluation of cationic polymers in RNAi-based cancer studies may offer a promising solution to this problem. In this chapter, we report the methodologies for comprehensive characterization of a biodegradable polymeric system for sh/siRNA delivery in cancer studies. The chapter will describe synthesis, characterization, and optimization of biodegradable poly (beta-amino ester) for sh/siRNA delivery. The protocols are provided for shRNA and siRNA complex preparation, stability and morphology study. Also, detailed methods are provided for the intracellular tracking and transfection of sh/siRNA using polymeric carrier. In addition, step-wise information is provided for the in vitro silencing of oncoprotein to study important cancer properties, including proliferation, malignancy, and metastasis of cancer cells.