Current Pharmaceutical Design - Volume 14, Issue 34, 2008

Oligonucleotide-based therapeutic drugs of current interest include tools related to RNA interference (RNAi) such as short interfering RNA (siRNA), short hairping RNA (shRNA), or micro RNA (miRNA) as well as catalytic RNA and antisense oligonucleotides (asON). Based on resolved molecular patho-mechanisms of diseases and the causative involvement of genes in the development of diseases, their advantages include the mechanistic level of action, i.e. their ability to interfere with gene expression. On the technical level, challenges of the use of therapeutic oligonucleotide-based drugs include their delivery into target organs or tissues, their cellular uptake, the mode of their intracellular transport and their sub-cellular localization. All of those issues need to be successfully solved before their therapeutic application can be considered as sufficiently effective, i.e. before target suppression can be achieved at appropriate expense and realistic doses of the drug. Meanwhile it is obvious that the cellular delivery and the intracellular trafficking of oligomeric nucleic acid-based tools represent major technical hurdles for their successful therapeutic application. We feel that major new mechanistic insights are necessary before those steps of intracellular translocation of siRNA and/or components of RISC can be identified that are limiting in case of insufficient siRNA-induced target suppression. The cellular uptake and intracellular transport pathways as well as their underlying mechanisms need to be revealed before the technical hurdle of limited delivery and intracellular release can be conceivably addressed on a more rational basis. While the mode of action of various classes of transfectants and non-viral vector systems for oligonucleotide-based drugs seems to be largely enlightened, there is not much known on their intracellular fate. As a consequence, more quantitative methodology needs to be established in order to monitor trans-membrane transport as well as intracellular localization and mobility of siRNA as a function of their mode of delivery. Most promising approaches seem to be related to the intersection of molecular medicine, cell biology, biochemistry, biophysics, and chemistry. This view is exemplified by the collection of articles in this special issue which presents some of those interdisciplinary approaches. It provides a summary of major technologies related to the efficient cellular delivery of nucleic acid-based therapeutics. It also summarizes new methodology and new biological insights underlying the cellular and sub-cellular transport of oligonucleotides aiming their therapeutic application in vivo. Successful pre-clinical and clinical studies in the use of oligonucleotide-based therapeutics including asON and siRNA crucially rely on efficient delivery into target cells and target tissues. A comprehensive introduction into the various options for the cellular delivery of chemically synthesized siRNA by non-viral vector systems which include encapsulation, complex formation and conjugation of the cargo/siRNA is provided by Aigner in the first article of this special issue [1] who further includes an up-to-date list of in vivo studies in the use of formulated siRNA. Modes of administration of siRNA range from systemic and intravenous delivery to intra-tumoral, intra-thecal, nasal, and other local and applications. While systemic administration is commonly believed to deliver siRNA to a few sites and organs in vivo, local administration is thought to provide greater chances for a successful therapeutic delivery. One such example is the lung to which oligonucleotide-based drugs can be locally and topically delivery by nasal administration. Further, the lung as a target organ provides a very large and easily accessible surface for topical delivery of asON and siRNA and it is noteworthy that in a number of cases the use of transfectants could be omitted. Biological and technical characteristics of this target organ are summarized by Moschos et al. [2] who also provide a summary of past and ongoing in vivo studies in the use of asON and siRNA.

RNAi interference (RNAi) is an almost standard method for the knockdown of any target gene of interest in vitro, exploring a naturally occurring catalytic mechanism. Beyond functional analyses, the downregulation of pathologically relevant genes which are aberrantly expressed in a given disease will offer novel therapeutic approaches, also with regard to otherwise ‘undruggable’ genes. RNAi is mediated by small interfering RNAs (siRNA), and thus siRNA delivery in vivo is of critical importance for its implementation. Due to the instability and physicochemical properties of siRNAs, the development of strategies and formulations for siRNA protection, cellular uptake, correct intracellular localization and endosomal release, in combination with favourable pharmacokinetic properties, preferential delivery to the target organ, high biocompatibility and absence of unwanted side effects is crucial for the success of RNAi-based therapeutics. Approaches include the encapsulation in lipids, the complex formation with a variety of liposomes or cationic polymers, the chemical conjugation of siRNAs for example to peptides, aptamers or antibodies as well as other formulations. This review discusses non-viral strategies, based on different siRNA formulations and various modes of administration, for the delivery of therapeutic siRNAs to induce RNAi in vivo. It gives a comprehensive overview including a detailed listing of in vivo studies which have successfully employed various strategies for analytical or therapeutic siRNA-mediated gene targeting in different animal models, and presents a more in-depth description of some promising approaches with a special emphasis on polymers.

The accessibility to topical administration through inhalation, combined with its large surface area, has led to speculation that the lung might offer an ideal target for the application of oligonucleotide based therapeutics. In this review, we shall critically examine the challenges facing antisense and siRNA based approaches for target validation in vivo and as potential therapeutics. In particular, we shall discuss the antisense and siRNA based approaches in relation to factors such as delivery, distribution, stability, off-target effects, unwanted immune responses and the selection of the optimum mRNA targets.

Classical pharmaceutical research and development relies on the identification and validation of i) membranepermeable small molecules or ii) extracellular targets that are able to interfere with cellular key signaling pathways. During the last two decades the direct intracellular delivery of macromolecules developed into a novel paradigm to manipulate cell functions. This technique, commonly referred to as protein transduction, is based on the ability of certain peptides, designated as cell penetrating peptides (CPPs), to cross the cell membrane and deliver cargos such as peptides and proteins. Although protein transduction has been widely applied the process of cellular uptake remains poorly understood and therapeutic applications are rare thus far. The mechanism by which CPP-modified proteins adhere to, and cross the plasma membrane of cells as well as the subsequent intracellular trafficking is currently extensively investigated. While there is consent that CPP-cargos are internalized via endocytotic pathways the actual membrane translocation mechanism remains unclear. Recent studies indicate that CPP-cargo trapped in endosomal vesicles can be released into the cytosol by direct membrane perturbation or by retrograde delivery as demonstrated for various toxins. The present review seeks to outline the current state-of-the-art concerning the mechanism of protein transduction. A further mechanistic understanding will be critical to design transduction strategies that may open the door for cell-impermeable compounds to become intracellular effectors.

Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments.

One of the major challenges for new therapeutics molecules to enter the clinic remains improving their bioavailability and cellular uptake. Therefore, delivery has become a key stone in therapeutic development and several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs) or protein transduction domain (PTD). PTDs or CPPs were discovered twenty years ago, based on the potency of several proteins to enter cells and nowadays, numerous peptide carriers have been described and successfully applied for ex vivo and in vivo delivery of varying therapeutic molecules. Two CPP-strategies have been reported; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization and the second is based on the formation of stable complexes with drugs depending on their chemical nature. Peptide-Based-Nanoparticle Devices (PBND), correspond to short amphipathic peptides able to form stable nanoparticles with proteins and/or nucleic acids. Three PBND-families, PEP, MPG and CADY have been described, these carriers mainly enter cells independently of the endosomal pathway and efficiently deliver cargoes in a large variety of challenging cell lines as well as in animal models. This review will focus on the structure/function relationship of the PBND: CADY, PEP and MPG, in the general context of drug delivery. It will also highlight the requirement of primary or secondary amphipathic carriers for in vitro and in vivo delivery of therapeutic molecules and provide an update of their pre-clinical evaluation.

The phosphorothioate(PS)-stimulated cellular uptake of naked short interfering RNA (siRNA) into mammalian cells indicates a promising new mechanistic strategy because it makes use of a caveosomal, rather than an endosomal pathway, which is used by the majority of known delivery systems. This PS-stimulated mode delivers large amounts of siRNA primarily into the perinuclear space which is related to measurable though moderate target suppression. The observed limited efficacy seems to be related to intracellular trapping of siRNA. Here, we studied the intracellular localisation of siRNA and Argonaute 2 (Ago2), the major component of the RNA interference (RNAi) machinery, by density gradient centrifugation and fluorescence microscopy after PS-stimulated delivery or transfection with Lipofectamine 2000. The two cell lines ECV-304 and SKRC-35 both take up siRNA in the PSstimulated mode but only ECV-304 shows RNAi, i.e. siRNA-mediated suppression of lamin A/C expression, whereas SKRC-35 does not. This lack of RNAi in the latter cell line seems to be due to a block of an intracellular siRNA translocation process. This study provides strong evidence for the view that co-localisation of siRNA and Ago2 in the vicinity of the rough endoplasmic reticulum (rER) in ECV-304 cells is related to target inhibition, whereas density gradient fractionation of cell organelles shows a lack of co-localisation in SKRC-35 cells in which RNAi does not occur after the PS-mediated delivery. In summary, we propose to exploit this dual cell system to identify important steps of intracellular trafficking of siRNA after PS-mediated delivery that are crucial for its biological activity and which seem to be of general importance for the understanding of the intracellular trafficking and release of siRNA.

RNA interference (RNAi) is an evolutionary conserved post-transcriptional gene silencing mechanism, in which double stranded RNA effector molecules trigger the degradation of complementary mRNA transcripts. The use of RNAi to reduce gene expression with high specificity and ready availability is a powerful tool for reverse genetics and provides great therapeutic potential for targeting diseases caused by the expression of a deleterious gene or mutant allele, e.g. cancer and viral infections. Besides the known preferences of the RNAi technique, there is a need for the development of improved small double stranded silencing triggers with long lasting silencing activity and maximum specificity. The introduction of chemically modified nucleotides into short interfering RNAs (siRNAs) is currently the method of choice. In this review, we summarize the effects of various modifications on siRNA sub-cellular localization and silencing activity, discuss ideal chemical modifications and positions within siRNAs suited for their use in medical therapies and present a new perspective to study siRNA mediated silencing in vivo by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) to further improve RNAi-based pharmaceuticals.

The prospect of introducing siRNA in a cell, to induce silencing of the corresponding gene, has encouraged research into RNAi-based therapeutics as treatment for human diseases. At present, the siRNA molecules that are in a more advanced stage of clinical evaluation have a common factor: all are delivered locally at the site of the disease. Thus, the state of the art in delivery of siRNA appears to be the local administration. This can certainly be attributed to the characteristics of siRNA molecules, such as relatively high molecular weight, negative charge, and susceptibility to nuclease degradation, which make systemic application as a drug molecule difficult. When focusing on local administration, the main concerns for siRNA delivery can be restricted to the trafficking of siRNA molecules from the vicinity of the target cells, to the intracellular compartment where RNAi takes place, i.e. the cytoplasm. This contribution is focused on the barriers and challenges in trafficking of siRNA upon local delivery. First, an overview is given on the current state of the art for siRNA delivery in clinical trials. Second, recent successful preclinical studies, involving direct and local administration of siRNA, are reviewed. Third, emphasis is given to the endosomal escape. Some of our recent work is presented: the application of photochemical internalization (PCI) to improve the endosomal escape of siRNA lipoplexes in vivo. Finally, concluding remarks focus on the advantages of employing a technique such as PCI to enhance the endosomal escape of siRNA molecules.