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European Biopharmaceutical Review

Express Delivery

The ability of siRNAs to degrade any target RNA of choice has helped to launch RNA interference as a powerful new tool for many scientifi c disciplines and has led to innovative siRNA technologies in medicine and agriculture.

The term RNA interference refers to a process by which RNAs can undergo sequence-specifi c degradation, resulting in the suppression of transcripts which possess complementary sequences. Small interfering RNAs (siRNAs) play an integral role in RNA interference, and encompass a series of cellular pathways which include a defence strategy against invading pathogens as well as an inherent mechanism for the regulation of gene expression.

The recent discovery and identifi cation of siRNAs has prompted the generation of a new discipline in molecular biology, and has led to the development of siRNA technology as a powerful new tool by which to alter expression of specifi cally targeted genes. siRNA technology has rapidly moved forward from in vitro studies to the cell culture stage. At present, R&D has progressed to in vivo applications for siRNA technology, for uses as diverse as therapeutics against a variety of diffi cult to treat diseases including cancer and Alzheimer’s, and even to uses in plant science. A major challenge to be addressed in this undertaking is the development of effective in vivo siRNA delivery vehicles.

siRNA belongs to a class of double-stranded (ds)RNA molecules of approximately 20-25 nucleotides in length which contain 2-nucleotide 3’ overhangs at each terminus. The function of siRNA in post-transcriptional gene silencing has been elucidated; sequence-specifi c siRNAs prevent expression of genes which correspond to their sequence by either inhibiting

or destroying the mRNA that they encode. This mechanism appears to be universal for a broad spectrum of organisms. The RNA in question (be it an miRNA product from the cell itself or from an invading pathogen) becomes cleaved by a cellular endonuclease known as Dicer into short fragments of approximately 21 base pairs in length. siRNAs generated from Dicer are then incorporated into a multi-protein complex known as the RISC (RNA induced silencing complex). The RISC selectively degrades one strand of the siRNA. The remaining strand of siRNA (the guide strand) becomes part of a siRNA/RISC complex, and can base pair with and cleave any complementary RNA species. RNAs which possess complementarity to the siRNA present in this complex are prevented from being used as a template for protein synthesis (1,2). As a result, the gene corresponding to this particular RNA species becomes silenced.

In Vivo Delivery of siRNAs

The ability of artificially designed siRNAs to down-regulate expression of any gene of interest based upon its target sequence, coupled with the ease of siRNA transfection into both plant and mammalian cell culture, has enabled siRNA technology to become a powerful tool in both basic and applied research (3). For example, siRNAs have been extensively utilised in reverse genomics studies to identify the function of an unknown gene in a biological pathway. In a similar fashion, siRNAs can be employed to silence genes which play a specific role in diseases such as cancer. siRNAs have even been used to change a crop phenotype, or prevent an insect from acting as a vector for an infectious disease.

Without question, cell culture has been at the focal point of siRNA R&D. The application of siRNA technologies for delivery into living organisms has proven to be a challenge to say the least. One significant obstacle that has surfaced repeatedly for in vivo siRNA technology is the requirement for more effective delivery systems. Current problems include variations in siRNA stability in different cell types, toxicity of the delivery vehicle and immune response or adverse effects by the host, and the unintended co-suppression of additional, off-target sequences (by which a gene with a coincidentally similar sequence to the targeted gene may also be suppressed). Additional challenges include the rapid excretion of siRNA from the body prior to it performing its function, and the accumulation of siRNA into unintended body tissues.

Tomorrow’s siRNA Delivery Vehicles

Safe, accurate and efficacious siRNA delivery vehicles are currently under development. Efforts are being made to increase siRNA stability, decrease toxicity and adverse effects to the organism in general, while at the same time improving target cell uptake, intracellular trafficking and release into the cytoplasm. Increasing the stability of siRNA and reducing its vulnerability to nucleases is one challenge that has been met with considerable success. Since ribonucleases present in the blood can quickly degrade siRNA in its native form, a number of chemical modifications have been introduced which increase in vivo stability of siRNA, its resistance to nucleases and the retention of siRNAs in the body.

Besides direct modification to the chemical structure of siRNA, the formation of siRNA conjugating with other stable compounds has proven to be successful. For example, siRNAcholesterol conjugates have greatly improved the efficiency of siRNA uptake and internalisation into cells, thus dramatically improving silencing efficacy in in vivo animal models (4). Alternatively, a-tocopherol (vitamin E) has been attached to the 5’end of a siRNA targeting Apo B (5). a-tocopherol is released from the siRNA in the cytoplasm and the siRNA worked efficiently, causing a significant decrease in blood cholesterol levels. siRNA has also been conjugated to aptamers with a fair degree of success. An aptamer specific for the HIV-1 envelope glycoprotein delivered siRNAs to HIV infected cells, resulting in suppression of viral infection (6,7).

siRNA can be delivered by viral expression vectors or by nonviral, nanoliposome-based carriers (see Figure 1a and 1b). Both delivery systems have advantages and disadvantages, and are detailed in the following section.

Virus Expression Vectors

siRNA has been effectively carried and expressed by lentivirus expression vectors derived from HIV. HIV-derived lentivectors have been designed to stably incorporate siRNAs or transgenes into humans to down-regulate gene expression on a permanent, rather than transient basis, thus creating a means by which to provide gene therapies for cancers and other diseases (see Figure 1b). Since lentivirus vectors are engineered to be replication-incompetent and in fact contain no open reading frames of the original virus, they are considered to be very safe. At this point, a lentiviral vector has been demonstrated to silence green fluorescent protein (GFP) in mice, proving its potential as a siRNA delivery vehicle. Furthermore, when eggs from GFP-expressing transgenic mice were transduced with lentivirus-expressing siRNAs corresponding to GFP, reduced fluorescence could be seen not only in embryos but in the F1 pups (8,9). One drawback to the use of these vectors stems from the fact that insertional mutagenesis resulting from uncontrolled incorporation of the provirus into the host genome may cause activation of a proto-oncogene. Other viral expression vectors that are currently utilised as siRNA delivery systems include adenovirus and herpes simplex virus type 1. While siRNA delivery via virus expression vectors are able to provide tissue-specificity and high efficiency, issues common to the employment of virus expression vectors, such as unwanted immune responses by the host, remain and can pose significant problems.

Non-Viral Delivery Vehicles

Nanocarriers are synthetic polymers a few hundred nanometres in size and are under rapid development as delivery vehicles for siRNAs. siRNA nanocarriers are highly versatile, safe, and can bind to and enter specific target cells efficiently, undergoing biodegradation and releasing their siRNA payload. Some nanocarriers have been engineered as vehicles for local delivery and slowly release a particular drug or siRNA over a prolonged period of time (10).

Nanocarriers can include liposomes, micelles, emulsions and solid lipid nanoparticles. To maximise efficiacy and minimise toxicity, lipid nanocarriers have been optimised with regard to lipid composition, siRNA-to-lipid ratio and particle size (10). Neutral, hydrophilic polymers such as polyethylene glycol have been added to the outer surface of the nanoliposomes to increase their half-life as they circulate in the body. Other compounds such as protamines, cationic polypeptides rich in arginine residues, neutralise and assist in siRNA delivery, and can target siRNAs to specific tissue types. The Arg-Gly- Asp (ARG) peptides have been successfully used to target nanoparticles containing siRNAs to tumours (11). Antibodies have also been used to selectively target nanoparticles to specific cell types (12).

Local versus Systemic Administration of siRNAs

siRNA carriers are required for both systemic and local administration routes. siRNA has been used in clinical trials through direct application to the eye, skin, lung and muscle tissue. Targets that cannot be localised, or are inaccessible by local routes, such as metastatic or haematological cancers, require systemic siRNA delivery, including oral, intravenous or intraperitoneal administration. There are advantages and disadvantages to each route. Local administration requires lower doses of siRNA and results in fewer side effects. Systemic administration requires that the delivery vehicle must remain biologically active in the bloodstream for longer time periods, and also must have the additional property of being able to permeate barrier organs to reach its target tissue.

Applications of siRNA Technology in Medicine

Applications for siRNA technologies in medical research are many and diverse (13). siRNAs have been used to combat virus infection, cancer, and even a variety of neurodegenerative diseases. For example, siRNA technology can be utilised to treat chronic hepatitis B or C infection and even HIV/AIDS. Up to now, current antiviral drugs can only be used in a restricted fashion due to issues such as toxicity, cost and virus resistance to the drug. siRNAs are able to block infection by binding to specific viral sequences and targeting them for degradation by the host cell’s silencing pathway. siRNAs have also been developed to improve the safety of live vaccines. For example, insertion of a neuron-specific siRNA into the poliovirus genome can restrict its tissue tropism, yielding an attenuated vaccine that is less likely to replicate in the central nervous system. This additional property reduces the pathogenicity and improves the safety of the attenuated virus used as a live vaccine.

siRNA technology has also shown great promise to combat cancer, and several siRNA-based anticancer therapies are currently undergoing clinical trials. siRNAs have been designed to target a number of genes which are overexpressed in different tumour types. Therapeutic target genes such as the kinesin spindle protein (KIF11) or polo-like kinase 1 have been used in nanodelivery vehicles. Introduction of siRNAs by these methods led to a block in cell cycle progression and slowed down tumour growth in animal models.

siRNA technologies may also prove useful for slowing the progression of neurodegenerative diseases and improving the quality of life for individuals who suffer from these forms of illness. siRNAs or expression vectors which encode siRNA have been demonstrated to be capable of crossing the blood/ brain barrier to treat neurological disorders. For example, beta amyloid cleaving enzyme type 1, a protease important in the pathogenesis of Alzheimer’s disease, has been down-regulated in various regions of the brain through employment of siRNA technology, and beneficial effects with respect to memory improvement and cognitive function have been demonstrated.

Conclusion

siRNAs have proven to perform in a wide range of applications, from human diseases to plant pathogen resistance. A number of stumbling blocks which affect their pharmacological properties in vivo are currently being addressed, including sensitivity to nuclease degradation, toxicity to the host, short retention time in the circulatory system, uptake into target cells and release into the cytosol. A number of solutions used together under optimal conditions will resolve these problems and make siRNA technology a viable innovation for the future.

References

  1. Hannon G and Rossi J, Unlocking the potential of the human genome with RNA interference, Nature 431(2): pp371-308, 2004
  2. Elbashir S, Lendeckel W and Tuschl T, RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev 15(2): 188-200, 2001
  3. Min Suk Shim and Young Jik Kwon, Efficient and targeted delivery of siRNA in vivo, FEBS Journal 277(23): pp 4,814-4,827, 2010
  4. Wolfrum C et al, Mechanisms and optimization of in vivo delivery of lipophilic siRNAs, Nat Biotechnol 25: pp1,149-1,157, 2007
  5. Nishina K, Unno T, Uno Y, Kubodera T, Kanouchi T, Mizusawa H and Yokota T, Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol, Mol Ther 16(4): pp734-740, 2008
  6. Zhou J, Li H, Li S, Zaia J and Rossi JJ, Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy, Mol Ther 16(8): pp1,481-1,489, 2008
  7. Zhou J, Swiderski P, Li H, Zhang J, Neff CP, Akkina R and Rossi JJ, Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells, Nucleic Acids Res 37(9): pp3,094-3,109, 2009
  8. Tiscornia G, Singer O, Ikawa M and Verma MI, A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA, Proc Natl Acad Sci USA 100(4): pp1,844-1,848, 2003
  9. Mäkinen PI, Koponen JK, Kärkkäinen AM, Malm TM, Pulkkinen KH, Koistinaho J, Turunen MP and Ylä-Herttuala S, Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain, J Gene Med 8: pp433-441, 2006
  10. Buse J and El-Aneed A, Properties, engineering and applications of lipid-based nanoparticle drug-delivery systems: current research and advances, Nanomedicine (Lond) 5(8): pp1,237-1,260, 2010
  11. Schiffelers RM et al, Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle, Nucleic Acids Res 32: e149, 2004
  12. Pirollo KF et al, Materializing the potential of small interfering RNA via a tumor-targeting nanodelivery system, Cancer Res 67: pp2,938-2,943, 2007
  13. Hefferon KL, Innovations in siRNA research: a technology comes of age, Recent Pat Antiinfect Drug Discov 5(3): pp226-239, 2010

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Kathleen Hefferon received her PhD in Molecular Virology at the University of Toronto, Canada. She worked as a postdoc at the Boyce Thompson Institute for Plant Research and later as the Director of the Human Metabolic Research Unit in the Division of Nutritional Science at Cornell University. Most recently, Kathleen has written two books on agricultural biotechnology and is currently teaching Virology at the University of Toronto. Email: kathleen.hefferon@utoronto.ca

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