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Advanced Drug Delivery Reviews (v.59, #2-3)

Editorial Board (pp. ii).

RNAi therapeutics: Principles, prospects and challenges by Lars Aagaard; John J. Rossi (pp. 75-86).
RNA interference (RNAi) was discovered less than a decade ago and already there are human clinical trials in progress or planned. A major advantage of RNAi versus other antisense based approaches for therapeutic applications is that it utilizes cellular machinery that efficiently allows targeting of complementary transcripts, often resulting in highly potent down-regulation of gene expression. Despite the excitement about this remarkable biological process for sequence specific gene regulation, there are a number of hurdles and concerns that must be overcome prior to making RNAi a real therapeutic modality, which include off-target effects, triggering of type I interferon responses, and effective delivery in vivo. This review discusses mechanistic aspects of RNAi, the potential problem areas and solutions and therapeutic applications. It is anticipated that RNAi will be a major therapeutic modality within the next several years, and clearly warrants intense investigation to fully understand the mechanisms involved.

Keywords: siRNA; shRNA; RNAi; RNA interference; Antisense; miRNA; RISC


Gene manipulation through the use of small interfering RNA (siRNA): From in vitro to in vivo applications by Lekha Dinesh Kumar; Alan R. Clarke (pp. 87-100).
The conventional approach to investigate genotype–phenotype relationships has been the generation of gene targeted murine strains. However, the emergence of RNAi technologies has opened the possibility of much more rapid (and indeed more cost effective) genetic manipulation in vivo at the level of the transcriptome. Successful application of RNAi in vivo depends on intracellular targeted delivery of siRNA/shRNA molecules for efficient knockdown of the desired gene. In this review, we discuss the rationale and different strategies of using siRNA/shRNA for accomplishing the silencing of targeted genes in a spatial and /or temporally regulated manner. We also summarise the steps involved in extending these approaches to in vivo applications, with a specific focus upon the development of silencing in the mouse.

Keywords: siRNA; shRNA; Transgenic; Transfection; Target validation; Lentiviral; Adenoviral; Therapeutic; siRNA design; Methods of delivery; Mammalian system


Therapeutic potential for microRNAs by Christine C. Esau; Brett P. Monia (pp. 101-114).
MiRNAs are a conserved class of non-coding RNAs that negatively regulate gene expression post-transcriptionally. Although their biological roles are largely unknown, examples of their importance in cancer, metabolic disease, and viral infection are accumulating, suggesting that they represent a new class of drug targets in these and likely many other therapeutic areas. Antisense oligonucleotide approaches for inhibiting miRNA function and siRNA-like technologies for replacement of miRNAs are currently being explored as tools for uncovering miRNA biology and as potential therapeutic agents. The next few years should see significant progress in our understanding of miRNA biology and the advancement of the technology for therapeutic modulation of miRNA activity.

Keywords: microRNA; Antisense oligonucleotides; siRNA


Systemic siRNA delivery via hydrodynamic intravascular injection by David L. Lewis; Jon A. Wolff (pp. 115-123).
The main barrier to the use of RNAi in mammalian systems is the difficulty in delivering siRNA or shRNA to the appropriate tissues. Although progress has been made in this area, many of the technologies developed require specialized expertise and reagents that are beyond the reach of most investigators. In contrast, the hydrodynamic injection technique is simple to perform and enables highly efficient delivery of naked, unmodified siRNA to a number of tissues, especially the liver. This review describes the development of the technique and explores the possible mechanisms that enable uptake of siRNA to biological effect. Examples of the use of hydrodynamic injection in animal models of disease and for the study of gene function are presented and discussed.

Keywords: siRNA; Hydrodynamic injection; Gene knockdown


Non-viral siRNA delivery to the lung by Mini Thomas; James J. Lu; Jianzhu Chen; Alexander M. Klibanov (pp. 124-133).
SiRNAs exert their biological effect by guiding the degradation of their cognate mRNA sequence, thereby shutting down the corresponding protein production (gene silencing by RNA interference or RNAi). Due to this property, siRNAs are emerging as promising therapeutic agents for the treatment of inherited and acquired diseases, as well as research tools for the elucidation of gene function in both health and disease. Because of their lethality and prevalence, lung diseases have attracted particular attention as targets of siRNA-mediated cures. In addition, lung is accessible to therapeutic agents via multiple routes, e.g., through the nose and the mouth, thus obviating the need for targeting and making it an appealing target for RNAi-based therapeutic strategies. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Delivery of siRNAs relevant to lung diseases has been attempted through multiple routes and using various carriers in animal models. This review focuses on the recent progress in non-viral delivery of siRNAs for the treatment of lung diseases, particularly infectious diseases. The rapid progress will put siRNA-based therapeutics on fast track to the clinic.

Keywords: RNA interference; siRNA delivery; Lung


Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides by Bryan R. Meade; Steven F. Dowdy (pp. 134-140).
The cellular membrane constitutes an effective barrier that offers protection for the complex, yet highly ordered, intracellular environment that defines the cell. Passage of molecules across this barrier is highly regulated and highly restricted, with molecular size being the most significant criteria. Over the last 15 years, a class of small cationic peptides has been discovered that can defy the rules of membrane passage and can gain access to the intracellular environment. Importantly, cellular entrance is also permitted for covalently coupled cargo. The cationic nature of these peptides is crucial for their ability to bind and traverse the anionic cellular membrane. Cell penetrating peptides (CPPs) have been used for the delivery of a wide range of macromolecules including peptides, proteins and antisense oligonucleotides. With the recent advancement and understanding of RNA interference (RNAi), CPPs offer an attractive means for the cellular uptake of double-stranded siRNAs to induce a RNAi response. This review focuses on the potential use of CPPs to deliver siRNA into cells and the implications of this technology for both gene function determination and therapeutic potential.

Keywords: Exogenous siRNA delivery; Peptide transduction domain; Cell penetrating peptides


shRNA and siRNA delivery to the brain by William M. Pardridge (pp. 141-152).
The limiting factor in in vivo RNA interference (RNAi) is delivery. Drug delivery methods that are effective in cell culture may not be practical in vivo for intravenous RNAi applications. Nucleic acid drugs are highly charged and do not cross cell membranes by free diffusion. Therefore, the in vivo delivery of RNAi therapeutics must use targeting technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. For RNAi of the brain, the nucleic acid-based drug must first cross the brain capillary endothelial wall, which forms the blood–brain barrier (BBB) in vivo, and then traverses the brain cell plasma membrane. Similar to the delivery of non-viral gene therapies, plasmid DNA encoding for short hairpin RNA (shRNA) may be delivered to the brain following intravenous administration with pegylated immunoliposomes (PILs). The plasmid DNA is encapsulated in a 100 nm liposome, which is pegylated, and conjugated with receptor specific targeting monoclonal antibodies (MAb). Weekly, intravenous RNAi with PILs enables a 90% knockdown of the human epidermal growth factor receptor, which results in a 90% increase in survival time in mice with intra-cranial brain cancer. Similar to the delivery of antisense agents, short interfering RNAi (siRNA) duplexes can be delivered with the combined use of targeting MAb's and avidin–biotin technology. The siRNA is mono-biotinylated in parallel with the production of a conjugate of the targeting MAb and streptavidin. Intravenous RNAi requires the combined use of RNAi technology and a drug targeting technology that is effective in vivo.

Keywords: Blood–brain barrier; RNAi; Endothelium; Transferrin receptor; Insulin receptor; Monoclonal antibody


RNA interference and innate immunity by Mouldy Sioud (pp. 153-163).
RNA interference is an evolutionarily conserved gene silencing process triggered by double-stranded RNAs. Common to all cell types, is the production of 21–24 nucleotide small interfering RNA (siRNAs), which guide the RNA-induced silencing complex (RISC) to identify and cleave target mRNA sequences. Presently, this biological breakthrough method has revolutionised gene function studies and holds great promise as validating drug targets and treating human diseases. However, despite the success that has been achieved by this technology, studies carried in human blood cells have revealed that siRNAs could generate bystander effects, including the activation of innate immunity and inhibition of unintended target genes. Interestingly, 2’ uridine-modified siRNAs did not trigger TLR signalling, but they totally suppressed immune activation by immunostimulatory siRNAs when both molecules where delivered to the same endosomes. This review describes the recent advances in understanding the innate immune response to both single and double-stranded siRNAs. Also, it highlights the spectrum of molecular strategies allowing the design of therapeutic siRNAs with minimal side effects.

Keywords: RNA interference; Small interfering RNAs; Innate immunity; Toll-like receptors; 2′-ribose modifications; Off-target effects


Toxicogenomics of non-viral drug delivery systems for RNAi: Potential impact on siRNA-mediated gene silencing activity and specificity by Saghir Akhtar; Ibrahim Benter (pp. 164-182).
RNA interference (RNAi) is an evolutionary conserved cellular process for the regulation of gene expression. In mammalian cells, RNAi is induced via short (21–23nt) duplexes of RNA, termed small interfering RNA (siRNA), that can elicit highly sequence-specific gene silencing. However, synthetic siRNA duplexes are polyanionic macromolecules that do not readily enter cells and typically require the use of a delivery vector for effective gene silencing in vitro and in vivo. Choice of delivery system is usually made on its ability to enhance cellular uptake of siRNA. However, recent gene expression profiling (toxicogenomics) studies have shown that separate from their effects on cellular uptake, delivery systems can also elicit wide ranging gene changes in target cells that may impact on the ‘off-target’ effects of siRNA. Furthermore, if delivery systems also alter the expression of genes targeted for silencing, then siRNA activity may be compromised or enhanced depending on whether the target gene is up-regulated or down-regulated respectively. Citing recent examples from the literature, this article therefore reviews the toxicogenomics of non-viral delivery systems and highlights the importance of understanding the genomic signature of siRNA delivery reagents in terms of their impact on gene silencing activity and specificity. Such information will be essential in the selection of optimally acting siRNA-delivery system combinations for the many applications of RNA interference.

Keywords: Genocompatibility; siRNA; RNA interference; Gene silencing; Off target effects; Polymer genomics; Gene expression; Drug delivery; Carriers; Non-viral vectors
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