siRNA-based Therapeutics Research Articles

Nanovehicle-based Small Interfering RNA (siRNA) Delivery for Therapeutic Purposes: A New Molecular Approach in Pharmacogenomics.

RNA interference (RNAi) is a process for regulating the gene expression in which small interfering RNAs (siRNAs) silence target genes. siRNA-based therapy as a new molecular treatment approach, offers therapeutic prospects for many common diseases such as cancer and cardiovascular disorders. Nevertheless, the efficacy of siRNA delivery has, so far, remained a challenging issue. This is due to their easy degradation through the circulation system and the difficulties in the intracellular delivery to specific tissues where they silence the target genes. There have been many efforts to develop suitable, safe and effective siRNA delivery systems in the past decades. These efforts specifically aimed to protect siRNA from serum nucleases and deliver it to an intracellular region in the desired target cells. In this context, one of the new and popular approaches is nanovehicle-mediated siRNA delivery systems. Here, the authors reviewed and highlighted the recent advances in this exciting and fast growing field to help in the development of effective therapeutic tools in controlling human diseases. A literature search was conducted from electronic databases such as Pubmed and Google scholar including original articles and review articles. siRNA delivery systems potentially may be used in future medicine, particularly for untreatable or poorly treated diseases. As we learn more about these delivery systems, we can better use the tremendous opportunities provided by siRNA-based therapeutics. The results of ongoing clinical trials will play an important role in determining whether siRNA-based drugs can be considered as a new class of drugs.

Targeted Therapies for Pancreatic Cancer and Hurdles Ahead.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers with a median survival of 6 months after diagnosis. Intrinsic resistance to chemotherapeutics and lack of effective targeted therapies are the major factors contributing to dismal prognosis. Several important genetic alterations (i.e., mutations, deletions) have been identified to be involved in the initiation and progression of pancreatic cancer, including KRAS and inactivation of tumor suppressors, such as TP53, SMAD4 and CDKN2A. Unique tumor microenvironment with excessive stroma due to desmoplastic reaction is one of the major characteristics of PDAC, promoting tumor growth and leading to treatment failures. In addition, tumor stroma represents an important biological barrier for drug delivery and successful treatment of PDAC. Small interfering RNA (siRNA) has recently emerged as a potential and targeted therapeutic approach which is now evaluated in clinical trials. However, siRNA-based therapeutics face important challenges, including rapid serum degradation, poor tumor cell uptake and cellular uptake, leading to off-target effects. Therefore, there is a great need for the development of safe and effective nanoparticles for better tumor-specific delivery of anti-cancer therapeutics. In this article, the main challenges in the treatment of pancreatic cancer and recent advancements on nano delivery systems of chemotherapeutics and gene-targeted agents, used both in preclinical and clinical trials are reviewed.

Budding Alliance of Nanotechnology in RNA Interference Therapeutics

RNA interference (RNAi), as a novel technique in which RNA molecules limit or silence the gene expression, is currently a hot research topic for producing novel therapeutic materials for challenging diseases. In the development of RNAi-based therapies, nanoscale particles, with a varying diameter along with facile modification methods that can mediate effective RNAi with targeting potential, are gaining wide interest. The nanotechnology itself has tremendous potential in the field of healthcare, especially for the development of better pharmaceuticals. Nano-enabled delivery has shown great success in the delivery of RNAi based therapeutics to specific locations in the body. Especially, siRNAs show great potential for use in nucleic acid therapeutics because of their potent and specific RNAi-triggering activity. This review summarizes the advanced nanocarriers such as solid lipid nanoparticles, gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, polymeric nanoparticles, nanotransformers and curdlan nanoparticles with special emphasis on various aspects of siRNA-based therapeutics.

Green Tea Catechin Dramatically Promotes RNAi Mediated by Low-Molecular-Weight Polymers.

Cytosolic delivery is the major challenge that limits the clinical translation of siRNA-based therapeutics. Although thousands of polymers have been developed for siRNA delivery, the efficiency–toxicity correlation is unsatisfactory. Here, we report a facile strategy to fabricate core–shell-structured nanoparticles with robust siRNA delivery efficiency. The nanoparticle is prepared by entropy-driven complexation of siRNA with a green tea catechin to yield a negatively charged core, followed by coating low-molecular-weight polymers to form the shell. This supramolecular strategy facilitates the polymers condensing siRNA into uniform nanoparticles. The nanoparticle specifically down-regulates target genes in vitro and in vivo, and efficiently attenuates chronic intestinal inflammation in an inflammatory bowel disease model. Notably, the highly efficient nanoparticles are applicable for various polymers with different topologies and chemical compositions, providing a versatile technique to break down the efficiency–toxicity correlation of cationic polymers. The proposed strategy in this study permits the development of a promising platform for polymer-mediated siRNA delivery.

Targeted siRNA delivery using aptamer‐siRNA chimeras and aptamer‐conjugated nanoparticles

The sequence-specific gene-silencing ability of small interfering RNA (siRNA) has been exploited as a new therapeutic approach for the treatment of a variety of diseases. However, efficient and safe delivery of siRNA into target cells is still a challenge in the clinical development of siRNA-based therapeutics. Recently, nucleic acid-based aptamers that target cell surface proteins have emerged as a new class of targeting moieties due to their high specificity and avidity. To date, various aptamer-mediated siRNA delivery systems have been developed to enhance the RNA interference (RNAi) efficacy of siRNA via targeted delivery. In this review, we summarize recent advances in developing aptamer-mediated siRNA delivery systems for RNAi therapeutics, mainly aptamer-siRNA chimeras and aptamer-functionalized nanocarriers incorporating siRNA, with a focus on their molecular designs and formulations. In addition, the challenges and engineering strategies of aptamer-mediated siRNA delivery systems for clinical translation are discussed. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Toward Understanding Liposome-Based siRNA Delivery Vectors: Atomic-Scale Insight into siRNA-Lipid Interactions.

Liposome carriers for delivering small interfering RNA (siRNA) into target cells are of tremendous importance because the siRNA-based therapy offers a completely new approach for treating a wide range of diseases, including cancer and viral infections. In this paper, we employ the state-of-the-art computer simulations to get an atomic-scale insight into the interactions of siRNA with zwitterionic (neutral) lipids. Our computational findings clearly demonstrate that siRNA does adsorb on the surface of a neutral lipid bilayer. The siRNA adsorption, being rather weak and unstable, is driven by attractive interactions of overhanging unpaired nucleotides with choline moieties of lipid molecules. It is the presence of the unpaired terminal nucleotides that underlies a drastic difference between siRNA and DNA; the latter is not able to bind to the zwitterionic lipid bilayer. We also show that adding divalent Ca ions leads to the formation of stable siRNA-lipid system complexes; these complexes are stabilized by Ca-mediated aggregates of siRNA and lipid molecules rather than by the overhanging siRNA nucleotides. Furthermore, the molecular mechanism of interactions between siRNA and the lipid bilayer in the presence of divalent cations seems to involve exchange of Ca ions between the outer mouth of the major groove of siRNA and the lipid/water interface. Overall, our findings contribute significantly to a deeper understanding of the structure and function of liposome carriers used for siRNA delivery and can be used as a theoretical basis for further development of siRNA-based therapeutics.

Design and Delivery of Therapeutic siRNAs: Application to MERS-Coronavirus.

The MERS-CoV is a novel human coronavirus causing respiratory syndrome since April 2012. The replication of MERS-CoV is mediated by ORF 1ab and viral gene activity can be modulated by RNAi approach. The inhibition of virus replication has been documented in cell culture against multiple viruses by RNAi approach. Currently, very few siRNA against MERS-CoV have been computationally designed and published. In this review, we have discussed the computational designing and delivery of potential siRNAs. Potential siRNA can be designed to silence a desired gene by considering many factors like target site, specificity, length and nucleotide content of siRNA, removal of potential off-target sites, toxicity and immunogenic responses. The efficient delivery of siRNAs into targeted cells faces many challenges like enzymatic degradation and quick clearance through renal system. The siRNA can be delivered using transfection, electroporation and viral gene transfer. Currently, siRNAs delivery has been improved by using advanced nanotechnology like lipid nanoparticles, inorganic nanoparticles and polymeric nanoparticles. The efficacy of siRNA-based therapeutics has been used not only against many viral diseases but also against non-viral diseases, cancer, dominant genetic disorders, and autoimmune disease. This innovative technology has attracted researchers, academia and pharmaceuticals industries towards designing and development of highly effective and targeted disease therapy. By using this technology, effective and potential siRNAs can be designed, delivered and their efficacy with toxic effects and immunogenic responses can be tested against MERS-CoV.

Water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of siRNA

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40–80%, when tested in a panel of lung epithelial cells. Considering the formulation ‘developability’, there were no significant changes in the complexes’ size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.

Dual peptide-mediated targeted delivery of bioactive siRNAs to oral cancer cells in vivo

ObjectivesDespite significant advances in cancer treatment, the prognosis for oral cancer remains poor in comparison to other cancer types, including breast, skin, and prostate. As a result, more effective therapeutic modalities are needed for the treatment of oral cancer. Consequently, in the present study, we examined the feasibility of using a dual peptide carrier approach, combining an epidermal growth factor receptor (EGFR)-targeting peptide with an endosome-disruptive peptide, to mediate targeted delivery of small interfering RNAs (siRNAs) into EGFR-overexpressing oral cancer cells and induce silencing of the targeted oncogene, cancerous inhibitor of protein phosphatase 2A (CIP2A). Materials and methodsFluorescence microscopy, real-time PCR, Western blot analysis, and in vivo bioimaging of mice containing orthotopic xenograft tumors were used to examine the ability of the dual peptide carrier to mediate specific delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells/tissues. ResultsCo-complexation of the EGFR-targeting peptide, GE11R9, with the endosome-disruptive 599 peptide facilitated the specific uptake of siRNAs into oral cancer cells overexpressing EGFR in vitro with optimal gene silencing observed at a 60:30:1 (GE11R9:599:siRNA) molar ratio. Furthermore, when administered systemically to mice bearing xenograft oral tumors, this dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone and significantly enhanced CIP2A silencing. ConclusionHerein we provide the first report demonstrating the clinical potential of a dual peptide strategy for siRNA-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells.

Overcoming the Challenges of siRNA Delivery: Nanoparticle Strategies

Despite therapeutics based on siRNA have an immense potential for the treatment of incurable diseases such as cancers. However, the in vivo utilization of siRNA and also the delivery of this agent to the target site is one of the most controversial challenges. The helpful assistance by nanoparticles can improve stable delivery and also enhance efficacy. More nanoparticle-based siRNA therapeutics is expected to become available in the near future. The search strategy followed the guidelines of the Centre of Reviews and Dissemination. The studies were identified from seven databases (Scopus, Web of Science, Academic Search Premiere, CINAHL, Medline Ovid, Eric and Cochrane Library). Studies was selected based on titles, abstracts and full texts. One hundred twenty nine papers were included in the review. These papers defined hurdles in RNAi delivery and also strategies to overcome these hurdles. This review discussed the existing hurdles for systemic administration of siRNA as therapeutic agents and highlights the various strategies to overcome these hurdles, including lipid-based nanoparticles and polymeric nanoparticles, and we also briefly reviewed chemical modification. Delivery of siRNA to the target site is the biggest challenge for its application in the clinic. The findings of this review confirmed by encapsulation siRNA in the nanoparticles can overcome these challenges. The rapid progress in nanotechnology has enabled the development of effective nanoparticles as the carrier for siRNA delivery. However, our data about siRNA-based therapeutics and also nanomedicine are still limited. More clinical data needs to be completely understood in the benefits and drawbacks of siRNA-based therapeutics. Prospective studies must pay attention to the in vivo safety profiles of the different delivery systems, including uninvited immune system stimulation and cytotoxicity. In essence, the development of nontoxic, biocompatible, and biodegradable delivery systems for medical application of RNAi-based therapeutics is needed.

Guide Strand 3'-End Modifications Regulate siRNA Specificity.

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.

Solid nano-in-nanoparticles for potential delivery of siRNA

siRNA-based therapeutics possess great potential to treat a wide variety of genetic disorders. However, they suffer from low cellular uptake and short half-lives in blood circulation; issues that remain to be addressed. This work is, to the best of our knowledge, the first to report the production of solid nano-in-nanoparticles, termed double nano carriers (DNCs) by means of the innovative technology of nano spray drying. DNCs (with a median size of 580–770nm) were produced by spraying at low temperatures (50°C) to prevent damage to heat-sensitive biomacromolecules like siRNA. DNCs consisting of Poly (d,l-lactide-co-glycolide) used as a wall material, encapsulating 20% human serum albumin primary nanoparticles (PNPs) loaded with siRNA, were obtained as a dry nanoparticulate powder with smooth spherical surfaces and a unique inner morphology. Incubation of pegylated or non-pegylated DNCs under sink conditions at 37°C, elicited a controlled release profile of the siRNA for up to 12 or 24h, respectively, with a minimal burst effect. Prolonged incubation of pegylated DNCs loaded with active siRNA (anti EGFR) in an A549 epithelial cell culture monolayer did not induce any apparent cytotoxicity. A slow degradation of the internalized DNCs by the cells was also observed resulting in the progressive release of the siRNA for up to 6days, as corroborated by laser confocal microscopy. The structural integrity and silencing activity of the double encapsulated siRNA were fully preserved, as demonstrated by HPLC, gel electrophoresis, and potent RNAi activity of siRNA extracted from DNCs. These results demonstrate the potential use of DNCs as a nano drug delivery system for systemic administration and controlled release of siRNA and potentially other sensitive bioactive macromolecules.

Current development of targeted oligonucleotide-based cancer therapies: Perspective on HER2-positive breast cancer treatment.

This Review discusses the various types of non-coding oligonucleotides, which have garnered extensive interest as new alternatives for targeted cancer therapies over small molecule inhibitors and monoclonal antibodies. These oligonucleotides can target any hallmark of cancer, no longer limited to so-called "druggable" targets. Thus, any identified gene that plays a key role in cancer progression or drug resistance can be exploited with oligonucleotides. Among them, small-interfering RNAs (siRNAs) are frequently utilized for gene silencing due to the robust and well established mechanism of RNA interference. Despite promising advantages, clinical translation of siRNAs is hindered by the lack of effective delivery platforms. This Review provides general criteria and consideration of nanoparticle development for systemic siRNA delivery. Different classes of nanoparticle candidates for siRNA delivery are discussed, and the progress in clinical trials for systemic cancer treatment is reviewed. Lastly, this Review presents HER2 (human epidermal growth factor receptor type 2)-positive breast cancer as one example that could benefit significantly from siRNA technology. How siRNA-based therapeutics can overcome cancer resistance to such therapies is discussed.

Nanoparticle delivery systems for siRNA-based therapeutics.

RNA interference (RNAi) is a naturally occurring endogenous regulatory process in which the short double-stranded RNA causes sequence-specific post-transcriptional gene silencing. Small interfering RNA (siRNA) represents great therapeutic potential for diseases (e.g., cancer) caused by abnormal gene overexpression or mutation. The major challenge in using RNAi for disease therapy is the targeted and effective delivery of siRNA in vivo. The development of nanotechnology with the important progress in nanoparticle-mediated delivery systems promises efficient intracellular siRNA delivery. This review summarizes the recent advances in nanoparticle delivery systems for siRNA-based therapeutics.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Clinical experiences with systemically administered siRNA-based therapeutics in cancer.

Small interfering RNA (siRNA)-based therapies are emerging as a promising new anticancer approach, and a small number of Phase I clinical trials involving patients with solid tumours have now been completed. Encouraging results from these pioneering clinical studies show that these new therapeutics can successfully and safely inhibit targeted gene products in patients with cancer, and have taught us important lessons regarding appropriate dosages and schedules. In this Review, we critically assess these Phase I studies and discuss their implications for future clinical trial design. Key challenges and future directions in the development of siRNA-containing anticancer therapeutics are also considered.

Current and Future Advances in siRNA Drug Delivery Vehicles

RNA interference has experienced an exponential increase in research interest since it was first reported nearly 2 decades ago. However, due to difficulties in drug delivery techniques, siRNA-based therapeutics have not reached their fullest potential in the clinical setting. The field of nanotechnology aims at mitigating many of these hurdles through the design and implementation of novel and sophisticated drug delivery vehicles. This review illustrates some of the challenges faced by siRNA drug delivery and considers how new nanomedical approaches can circumvent these biological barriers.

Biomimetic gold nanocomplexes for gene knockdown: Will gold deliver dividends for small interfering RNA nanomedicines?

RNA interference (RNAi) effectors such as small interfering RNA (siRNA) and micro RNA (miRNA) can selectively downregulate any gene implicated in the pathology of a disease. Therefore, RNAi-based therapies have immense potential for the treatment of a wide range of diseases. However, pharmacokinetic and pharmacodynamic studies have revealed that these therapeutic agents have poor bioactivity due to a number of factors, including insufficient plasma drug levels, short plasma half-lives, renal clearance, and hepatic metabolism. Non-viral delivery may facilitate the clinical application of siRNA-based therapeutics by helping to overcome these barriers. Recently, the potential of gold nanoparticles (AuNPs) as multifunctional carriers for transporting drugs, proteins, and genetic materials has been demonstrated. In this review, some of the key properties of AuNPs relevant to siRNA delivery, such as physical properties and surface chemistry have been described. In addition, the ability of AuNP-based formulation strategies to successfully overcome delivery barriers associated with siRNA, and the potential for this material to translate into safe and effective nanomedicines are critically discussed.

Enhanced delivery of PEAL nanoparticles with ultrasound targeted microbubble destruction mediated siRNA transfection in human MCF-7/S and MCF-7/ADR cells in vitro.

The gene knockdown activity of small interfering RNA (siRNA) has led to their use as potential therapeutics for a variety of diseases. However, successful gene therapy requires safe and efficient delivery systems. In this study, we choose mPEG-PLGA-PLL nanoparticles (PEAL NPs) with ultrasound targeted microbubble destruction (UTMD) to efficiently deliver siRNA into cells. An emulsification-solvent evaporation method was used to prepare siRNA-loaded PEAL NPs. The NPs possessed an average size of 132.6±10.3 nm (n=5), with a uniform spherical shape, and had an encapsulation efficiency (EE) of more than 98%. As demonstrated by MTT assay, neither PEAL NPs nor siRNA-loaded PEAL NPs showed cytotoxicity even at high concentrations. The results of cellular uptake showed, with the assistance of UTMD, the siRNA-loaded PEAL NPs can be effectively internalized and can subsequently release siRNA in cells. Taken together, PEAL NPs with UTMD may be highly promising for siRNA delivery, making it possible to fully exploit the potential of siRNA-based therapeutics.

Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection.

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape.