Current Topics in Medicinal Chemistry - Volume 9, Issue 12, 2009

RNA interference (RNAi) is a recently realized technique that allows for the targeted control of gene expression. In some diseases genes are overtly regulated, thus the ability to specifically and potently target the silencing of gene expression has significant medical impact on these disease states. RNAi utilizes small interfering RNAs (siRNAs) or microRNAs (miRNAs) to silence specific genes. There are two modes of RNAi-based regulation in human cells: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). There are several nuances in the mechanistic underpinnings in both PTGS and TGS, which are discussed in this issue of “Current Topics in Medicinal Chemistry”. These articles provide a comprehensive review of recent developments in RNAi in human cells and highlight the distinct mechanisms in this state of the art technique. In this issue, articles from 8 research groups actively utilizing RNAi have been compiled to provided a thorough and comprehensive analysis of the current state of RNAi as a therapeutic tool. Dr. Marc S. Weinberg and colleagues opens the issue with an article entitled; “RNA interference therapy or another antisense modality”? In this article RNAi is discussed and mechanistically contrasted with antisense technologies which for several years were the dominant paradigm utilized in regulating gene expression. Indeed it was initial studies by Fire and Mello utilizing antisense RNAs which uncovered the RNAi pathway in C. Elegans. The second review by Dr. Kazsuz Suzuki and colleagues entitled; “Transcriptional targeted RNAs”, discusses the relatively new pathway of small RNA directed transcriptional silencing. This pathway involves small RNAs capable of directing silent state epigenetic changes at gene promoters, and recent investigation suggests that this pathway is endogenous in human cells and relies on long non-coding RNAs for functional gene regulation. Notably, this method of silencing can result in stable epigenetic silencing which can be long-term and passage to daughter cells. Far less is known regarding this molecular pathway but the potential of silencing may be superlative with regards to the well studies RNAi pathway.

The naturally-occurring RNA interference (RNAi) pathway represents a powerful tool for the sequence-specific post-transcriptional silencing of gene expression. By exploiting the endogenous mammalian RNAi pathway, several expression-based strategies have been developed to inhibit human immunodeficiency virus (HIV) gene expression and replication. This approach potentially has utility as a protective ‘therapeutic vaccine’ of virus-susceptible lymphocytes. In this review we discuss new developments aimed at improving efficacy and delivery of novel RNAi-based gene expression antiviral strategies. Particular attention is given to advances in combinatorial gene expression systems that prevent the emergence of RNAi-resistant virus by simultaneously targeting multiple HIV targets. Potential usefulness of silencing host factors that are required for viral replication is also discussed. These approaches form the basis for a number of promising ongoing and future clinical trials aimed at providing an effective, safe and prolonged single-intervention therapy for HIV/AIDS.

Small RNA molecules, including small interfering RNA (siRNA) and micro RNA (miRNA), have rapidly emerged as important regulators of gene expression. Recent articles have demonstrated RNA mediated complex induced transcriptional gene silencing (TGS) occurring in the nucleus. Originally the small RNA mediated TGS pathway has been reported in yeast and plants, currently a number of articles strongly suggest that this newly established gene silencing mechanism is present in mammals. RNA mediated TGS has been reported for various human promoters including inhibition of tumor susceptibility genes, X-chromosome inactivation and suppression of human chemokine receptor. Small RNAs can inhibit human viral infection through the TGS pathway. Prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the HIV-1 promoter has been reported initially using a susceptible adherent cell line model and recently prolonged suppression of productive HIV-1 infection in a T-cell line model has been demonstrated by a retrovirally delivered short-hairpin RNA (shRNA) targeting the same region. RNA mediated gene silencing in HIV-1 infection can induce heterochromatin (closed) structure in the promoter regions, which is consistent to those changes seen in studies of various RNA directed TGS in various human promoter regions. More recent observations suggest transcriptional activation can be induced through RNA duplexes targeting the human promoter of E-cadherin, p21 and the progesterone receptor. Although the precise mechanisms of how RNA mediated transcriptional gene silencing or activation functions has yet to be elucidated, this review describes linkage of small RNA mediated gene regulation and induction of epigenetic regulation in the promoter region in mammals.

Applying RNA interference to silence a specific gene has opened a new and promising avenue of gene therapy. But a key bottleneck is the poor stability and inability of naked siRNA to translocate through cell membranes. Among several delivery systems, cationic peptides capable of penetrating cell membranes have drawn attention due to their structural and functional versatility, potential biocompatibility and ability to target cells. In this review, different classes of peptides employed in siRNA delivery are reviewed. In particular, a new class of siRNA delivery peptides with high transfection efficiency and low cytotoxicity is introduced.

RNA interference (RNAi) is an attractive phenomenon for practical use that specifically inhibits gene expression and is carried out by small double-stranded RNAs (dsRNAs) including small interfering RNA (siRNA) or short hairpin RNA (shRNA). In addition, RNAi is of great interest for clinical use to cure refractory diseases related to the expression of a specific gene. To achieve gene silencing in the body, a sufficient amount of dsRNA must be delivered and internalized into target cells. However, dsRNAs have a large molecular weight and net negative charge, which limits their membrane-permeating ability. Moreover, dsRNAs are rapidly degraded by endonucleses in the body. Therefore, for the efficient delivery of dsRNAs, many approaches based on drug delivery systems have been carried out. In this review, we focus on recent reports about the application of functional peptides and proteins designed for the efficient delivery of dsRNAs.

The efficient delivery of biologically functional short interfering RNA (siRNA) in vivo remains a widely unresolved technical problem in therapeutic drug development. The repertoire of concepts for the cellular uptake of oligonucleotide-based tools and drugs has been extended by the mechanistically novel finding that phosphorothioate (PS)- modified single-stranded oligodeoxyribonucleotides (ON) promote the intracellular accumulation of naked extra-cellular siRNA in a variety of cell types. This mode of delivery gives rise to substantial intracellular amounts of siRNA, up to 104 siRNA molecules per cell. Conversely, the moderate biological effectiveness strongly indicates that intracellular release of siRNA from sub-cellular compartments where it seems to be trapped is a necessary step towards efficient target suppression. Here, we summarize key characteristics of the PS-stimulated cellular uptake of siRNA and describe concepts for the increase of intracellular delivery of biologically functional siRNA.

Synthetic small interfering RNAs (siRNAs) open promising new therapeutic perspectives in acute and chronic pathologies. A number of experiments in mice demonstrated the ability of naked siRNAs injected under a normal pressure to trigger gene silencing in vivo, translating into a measurable phenotype. We focus in this review on the information that we can gain from these experiments, and discuss how the specificity of the gene silencing in vivo can be controlled. Because the activity of most drugs increases with the dosing, we are prone to consider that increasing the concentration of siRNAs within cells enhances the efficiency and the duration of the silencing. However, because RNAi is a saturable process, and because increasing the siRNA concentration into cells can induce undesirable side effects, this must be demonstrated. We compare in this review the methods used to quantify and study the biodistribution of siRNAs in living animals, and discuss how these methods can help in designing for each model and each siRNA the most adequate protocol to silence a cognate target gene in vivo.

RNA interference (RNAi) holds great promise as gene therapy approach against viral pathogens, including HIV-1. A specific anti-HIV-1 response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular transgene expression of short hairpin RNAs (shRNAs) or microRNAs (miRNAs). Both targeting of the viral mRNAs or the mRNAs for cellular co-factors that are required for viral replication have been shown successful in suppressing HIV-1 replication. However, like conventional mono-therapies, the use of a single anti-HIV-1 RNAi inducer results in the emergence of RNAi-escape mutants. To prevent viral escape, a combinatorial RNAi approach should be used in which multiple RNAi effectors against HIV-1 are simultaneously expressed. Although induced RNAi is able to trigger a robust and specific knockdown of virus replication, it is becoming apparent that RNAi therapeutics encounter difficulties concerning off-target effects, cellular toxicity and specific delivery to the right cells. This review covers the recent progress in combinatorial RNAi-based approaches against HIV-1 using lentiviral vectors as a delivery system. The potential for a clinical gene therapy application will be discussed.

Aptamers that are evolved by the SELEX procedure (Systematic Evolution of Ligands by Exponential enrichment) can specifically recognize and tightly bind their cognate targets by means of well-defined secondary and three-dimensional structures. In comparison to antibodies, nucleic acid-based aptamers offer some exciting advantages, including the potential for chemical synthesis, convenient modification, chemical versatility, stability and lack of immunogenicity. During the past 20 years, aptamers have been developed for various applications such as diagnostics, drug development, target validation and therapeutics. Aptamers targeting cell surface proteins are being explored as promising delivery vehicles to target a distinct disease or tissue in a cell-type-specific manner. In this review, we summarize the recent developments in creatively using cell-internalizing aptamers as drug delivery escorts to deliver, enhance and modulate the activity of other therapeutic agents, including chemical drugs, toxins, small interfering RNAs and nanoparticle-encapsulated drugs. Specifically, several attractive aptamer-mediated cell-type specific siRNA delivery systems are highlighted, and their promise in clinical development is also discussed.