In the one decade that have passed since the discovery of RNA interference (RNAi), efforts and huge money have been invested in research to find out the therapeutic application of gene silencing in humans and the number of publications related to RNAi have also increased exponentially. There is encouraging data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. A significant advantage of this siRNA-based technology is the rapidity with which different siRNA sequences and the matching genes can be studied, particularly useful for drug target validation. However, the major challenge in navigating these molecules for regulatory approval is inefficient delivery to the target site resulting in serious off target effects and immunological responses. The widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. This review highlights the potential of siRNAs in gene silencing over other technologies, the various challenges encountered during in-vivo siRNA delivery with possible strategies to overcome them and a view to the prospect of siRNAs in therapeutics.
Gene therapy involves gene replacing, swapping, repairing or silencing of a nonfunctional or transmuted gene to treat diseases such as cancer, HIV, etc. Gene silencing means "switching off" of a particular gene by a mechanism other than genetic modification and can be achieved by distinct strategies viz. ribozyme technology, antisense oligonucleotides, aptamers and RNAi (figure 1). Within a short span of time, the use of RNAi has rapidly spread to nearly every aspect of biomedical research from target validation to therapeutics. siRNAs, shRNAs and the most recently discovered miRNAs are the functional mediators of RNAi.
siRNAs are double stranded RNA fragments generated by the cellular enzyme Dicer, typically comprising of 19 to 23 nucleotides with a molecular weight of approximately 13 to 15 kD and a negative charge of 38 to 46 mV.
Dicer processes dsRNAs into 21-25 nucleotide siRNAs which are subsequently incorporated into one or more of the Argonaute proteins in RNA-induced silencing complex (RISC) where the RNA serves as a sequence specific guide for complementary base pairing with the target and guides RISC for sequence specific target degradation or translational inhibition.
For gene silencing activity intracellular entry into the target cell within the diseased tissue and subsequent appearance in the cytoplasm to silence the target mRNA is required for the siRNAs. However, primary obstacles for achieving this in vivo include competitive uptake by nontarget cells, excretion in urine, degradation by nucleases, and endosomal trapping. Overwhelming efforts have been made and are still in progress for the development of promising siRNA therapeutics by chemical manipulations, designing appropriate delivery vector, using targeting ligands, or a combination thereof. This critique summarizes the potential of siRNAs as therapeutics, challenges in siRNA delivery along the possible strategies used to overcome these with a view to the future of siRNAs in the management of chronic diseases.
Why siRNAs?
siRNA based RNAi technology has been acknowledged by researchers for its therapeutic potential as a potent, highly specific and cost-effective way of treating diseases by inhibiting a target protein far before its formation before translation. The leeway of chemical modification and designed synthesis has opened enormous possibilities of using siRNAs as potential therapeutics. Most drugs provide only a symptomatic relief from a disease rather than destroying its root cause i.e. the disease causing protein. Even cell-specific drugs like monoclonal antibodies have limited targets and act mainly on circulating proteins or cell-surface receptors.
Compared to other antisense strategies siRNAs are highly specific efficiently blocking the production of the disease causing protein before it is translated. A potential aspect of siRNA therapeutics is that they can be precisely tailor made to access unlimited number of intracellular targets with the knowledge of sequenced human genome and delivery methodologies. RNAi is an innate biological response, and hence represents a more natural strategy for manipulating gene expression making use of siRNAs and miRNAs. Further, certain diseases are caused by mutation in a single allele with siRNAs targeting specifically the disease-causing mutation leaving the normal allele intact.
Compared to other antisense agents, siRNAs have longer half life and are effective at very small concentrations avoiding the need of repeated administrations. Most viral diseases are manifestations of changing viral mutations that limits the use of an antiviral drug against a particular mutant only. A blend of mutant specific siRNAs in a single delivery vector is a wonderful remedy for such mutating diseases. Furthermore, considering multi drug resistance associated with chemotherapeutic and antibiotic therapies, siRNAs can specifically inhibit the expression of resistance causing proteins.
Thus, siRNAs enjoy a sound position in antisense therapy being easy amenability to target specific designing, specific and potent with the ease of administration through various routes like pulmonary, ocular, nasal, oral, intracerebral, intramuscular, intravenous, intratumoral, etc. Table 1 summarises the key features of siRNAs and other gene silencing approaches. However, the major challenge to siRNA delivery in-vivo is inefficient delivery to the target site and can be acknowledged by better understanding of the various barriers encountered as per its path from the site of delivery to the site of action.
Challenges in siRNA delivery
Degradation by nucleases Following administration, the first biological barrier encountered by siRNAs is presented by the nuclease activity in the blood and tissues resulting in low gene silencing. Chemical modifications at the 2’-OH position of pentose sugars or 3’ half of the siRNA structure, and use of suitable delivery vectors can drastically improve the stability of siRNAs towards nucleases. The substitution of sulphur for oxygen in phosphorothioate and 2’-OH modifications like in locked nucleic acids (LNAs), peptide nucleic acids (PNAs), etc. improve biological stability of siRNAs towards nucleases. Further, the inclusion of 6-carbon sugar instead of ribose and 2’-F and 2’-OMe modifications and use of gapmers also prolong the biological half life of siRNAs in vivo. Furthermore, the formulation of lipoplexes and polyplexes protect siRNAs from degradation by nucleases.
Glomerular Filtration, Hepatic Metabolism and RES Uptake
Molecules less than 70kDa and 5nm in diameter undergo glomerular filtration and siRNAs being small readily get excreted soon after their administration. Also, following administration siRNAs easily get phagocytosed by the mature macrophages residing in the tissues of reticuloendothelial system (RES) like liver, spleen and lungs. While, particles smaller than 100 nm leak from the intercellular junction of capillary endothelium to the hepatic interstitial spaces due to hepatic uptake and get trapped by the Kupffer cells there.
Colloidal complexes of siRNA with cationic polymers or lipids get destabilized as aggregates due to the presence of serum proteins, platelets, and RBCs in the blood. Thus, both the size and charge of these complexes determine their clearance from the body. A coating of polyethylene glycol neutralises the surface charge and imparts a protective hydrophilic sheath around these complexes making them long circulating. Thus, clearance and RES uptake of siRNAs can be avoided by carefully manipulating the size and charge of the final complex to around 100nm and near to neutral respectively.
Endothelial barrier
The endothelial cells that line the vascular lumen present a barrier to siRNA delivery as need to cross the endothelium before being delivered to the tissue parenchymal cells. siRNAs being large and negatively charged can not readily cross the cellular membranes. However, certain tissues such as liver and spleen own relatively larger endothelial intercellular spaces than any other body tissue allowing access of even large siRNA molecules. They can follow either paracellular route through the intercellular pores, or the transcellular pathway which is claveolin based transcytosis, to egress across the endothelial lining. Cell penetrating peptides, targeting ligands or molecular conjugates can be used to facilitate passage of siRNAs across the endothelial lining.
Cellular uptake and subcellular distribution
For gene silencing activity siRNAs must undergo cellular uptake and sub cellular distribution to degrade the target mRNA in the cytoplasm. Transcellular pathway through receptor mediated endocytosis is the main mechanism of cellular uptake and can be facilitated by conjugating the siRNAs with a receptor specific ligand such as monoclonal antibodies or cell penetrating peptides like penetration, transportation, etc. After cellular uptake sequential intracellular trafficking into the subcellular compartments occurs and finally, siRNAs must be released from the endosomes to reach the target mRNA in the cytoplasm to effect gene silencing and is usually achieved by conjugating endosomal release signal peptides to siRNA entrapped within a nano carrier.
Immunological barrier
Our body has inbuilt mechanism to fight against invading foreign bodies conferred by two distinct mechanisms namely innate immunity and adaptive immunity and acts as a barrier to in vivo siRNA delivery. The innate immunity is of major concern in case of naked as well as viral vectors based siRNA delivery. Also, adaptive immunity mediated by T and B lymphocytes is of concern as interacts with specific surface receptors on T and B lymphocytes causing activation and production of effector T cells and antibodies against the invading foreign bodies. Researchers have reported serious immune responses with siRNA therapeutics.
Current status
Currently, various siRNA-based drugs are under clinical investigations (Table 2), although expressed short hairpins and at least one anti-miRNA antisense are in trials. The siRNAs in clinical trials are by far chemically synthesized, bypassing the Dicer cleavage step for entry into RISC. Only two clinical trials use ex vivo delivery, whereas most of the trials employ systemic delivery including injections directly into the target tissues such as the eyes for treatment of age-related macular degeneration or directly into tumours, inhalation or infusion of siRNAs incorporated in delivery vectors. However, there is no FDA approved siRNA drug till date, this may change within the next few years, and will open doors for more approved siRNA therapeutics.
Currently siRNAs are enjoying too many clinical applications for diseases. In clinics the safety assessment of siRNAs is more complex than classical drugs being an endogenous mechanism. However, there are no safety guidelines addressing siRNA till date with only a little information available from research engines.
Future envisaged
siRNAs have demonstrated promising results in early stages of clinical trials against mutating diseases. Today, siRNA based formulations are developed with patent point of view which is likely to boost investments in this arena. siRNAs use the endogenous mechanism of RNAi and we can expect maximal therapeutic benefit from siRNA delivery. Similarly, it can be used in almost all kind of diseases and can lead to personalized medicines. Many clinical trials have been initiated since last one decade after first report on siRNA use as therapeutics.
Pharma giants across the globe have joined hands to successfully deliver siRNAs in vivo and looking for glorious future. However, scenario in India is still gloomy and needs attention of Indian multinational pharmaceutical companies to collaborate with research/academic institutions to gear up the research in this rewarding area of research to reap the benefit of potential of siRNA in regular clinical practice for prevention, cure/biosensitization of available drugs in otherwise presently incurable diseases.
We expect to see siRNAs as standalone therapeutics in future, but also will be used in combination with current therapies for alleviation of human sufferings. Thus, a combined knowledge of human genome sequence, targeted delivery systems, and siRNA designed therapies will be used in future therapies including/against any mutating disease. Although, the systemic delivery of siRNA to specific targets and their availability in cytoplasm of pathogenic tissue still remains greatest challenge and needs attention of the drug delivery scientists.
Authors are faculty, Pharmacy Det, The Maharaja Sayajirao University, Baroda,Gujarat