Elimination Pathways of Systemically Delivered siRNA

The elimination process of systemically administered small interfering RNA (siRNA) was investigated by using siRNA labeled with an infrared fluorescent dye. A novel siRNA elimination pathway was identified. In this pathway, liver-enriched siRNA is secreted into the gallbladder and then emptied into the intestine. Blocking this pathway resulted in the absence of siRNA fluorescence within the intestine, with greatly enhanced siRNA accumulation in liver and gallbladder at the same time. Furthermore, we demonstrated that delivery carriers play an essential role in siRNA distribution and elimination, highlighting their importance in siRNA therapeutics.     

Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA

We recently reported that the prolonged circulation property of PEGylated cationic liposomes containing nucleic acids disappears, if the second dose is injected within a few days later, due to the production of anti-PEG IgM. This accelerated blood clearance is a concern for treating diseases which require repeated treatment with a PEGylated formulation containing nucleic acids. In this study, we investigated the effect of encapsulation of siRNA in a recently introduced PEGylated lipid nanocarrier for which the term “wrapsome” (PEGylated wrapsome, PEG-WS) was proposed as well as the sequence of the encapsulated siRNA on anti-PEG IgM production. siRNA encapsulated in PEG-WS produced little anti-PEG IgM relative to siRNA in conventional PEGylated lipoplexes. The sequence of siRNA in the PEG-WL dramatically affected the anti-PEG IgM production; a potent immune stimulatory siRNA induced a higher anti-PEG IgM production. Such enhanced effect was abrogated by incorporation of 2′-O-methyl (2′-OMe) uridine into the sequence of siRNA, probably via inhibiting cytokine induction such as IL-6 and TNF-α. Our results strongly indicate that the use of an encapsulation-type lipid nanocarrier with a low immuno-stimulatory siRNA may allow repeated dosing of siRNA containing PEGylated formulations without the induction of a strong immune reaction against PEG and thus may advance synthetic siRNA into a broad range of therapeutic applications.     

Innovative nanotechnologies for the delivery of oligonucleotides and siRNA.

One way to reach intracellular therapeutic targets in cells consists in the use of short nucleic acids which will bind specifically to on targets thanks to either Watson–Crick base pairing or protein nucleic acids recognition rules. Among these short nucleic acids an important class of therapeutic agents is antisense oligonucleotides and siRNAs. However, the major problem of nucleic acids is their poor stability in biological media. One method, among others, to solve the stability problem is the use of colloïdal carriers such as nanoparticles. Nanoparticles have already been applied with success to in vitro drug delivery to particular types of cells and in vivo in several experimental models. Many membrane and intracellular processes deal with nanosized structure (typically 100 nm) which are processed further through the recognition sites of receptors and enzymes. Thus non-viral nanoparticles are interesting candidates to present biochemical molecules such as nucleic acids and proteins to cells as well as to protect them in vivo during delivery. This review focuses on the recent developments in the design of nanotechnologies to improve the delivery of antisense oligonucleotides and siRNAs.     

Electroporation-enhanced gene delivery in mammary tumors

Electroporation was applied to enhance gene transfer into subcutaneous MC2 murine breast tumors. Cultured MC2 cells were also transfected by electroporation or by cationic liposomes in the presence of serum using pSV-luc plasmids. Electroporation parameters and liposome formulation were optimized to achieve the highest relative levels of transfection. An electric field threshold for successful electrotransfection in cultured cells appeared around 800-900 V/cm. The liposomes used contained the cationic lipid dioleoyl-3-trimethylammonium propane (DOTAP). Multilamellar vesicles (MLV) had a 10-fold advantage over small unilamellar vesicles (SUV) in cell culture transfection. For in vivo gene delivery, the plasmids were injected either alone, or in complex with MLV or SUV DOTAP liposomes. A series of six electric pulses 1 ms long were applied across tumors, using caliper electrodes on the skin surface. Electric field strengths ranged from 400-2300 V/cm. Luciferase expression was approximately two orders of magnitude higher than controls in tumors treated with pulses > or =800 V/cm. Differences between enhanced relative levels of transfection using uncomplexed plasmid and lipoplexes were not statistically significant. Distribution of DNA into tumor tissues was monitored by fluorescence in situ PCR. The highest numbers of fluorescent cells were found in tumors electroporated following the injection of plasmid. The significant transfection improvement shows that in vivo electroporation is a powerful tool for local gene delivery to tumors.     

Regulatory roles of extracellular vesicles in immune responses against Mycobacterium tuberculosis infection

Extracellular vesicles (EVs) are cystic vesicles naturally released by most mammalian cells and bacteria. EV contents include proteins, lipids, and nucleic acids. EVs can act as messengers to transmit a variety of molecules to recipient cells and thus play important regulatory roles in intercellular signal transduction. EVs, released by either a host cell or a pathogen, can carry pathogen-associated antigens and thus act as modulators of immune responses. EVs derived from Mycobacterium tuberculosis (Mtb)-infected cells can regulate the innate immune response through various pathways, such as regulating the release of inflammatory cytokines. In addition, EVs can mediate antigen presentation and regulate the adaptive immune response by transmitting immunoregulatory molecules to T helper cells. In this review, we summarize the regulatory roles of EVs in the immune response against Mtb.     

Incorporation of histone derived recombinant protein for enhanced disassembly of core-membrane structured liposomal nanoparticles for efficient siRNA delivery

A novel recombinant protein tetra-H2A (TH) derived from histone H2A has been developed to replace protamine as a conditionally reversible, nucleic acid condensing agent. The novel protein will address the insufficient release of nucleic acid therapeutics, which is captured by protamine for siRNA delivery. TH is composed of 4 tandem repeats of the histone H2A N-terminal sequence, intervened by the cathepsin D cleavage site. The repeating H2A sequence enhances the binding affinity to anionic nucleic acids, forming more stable condensates, as demonstrated by the binding affinity assay. The TH/siRNA condensates are formulated into a core-membrane structured liposomal nanoparticle (NP). The endosomes of cancer cells are rich in cathepsin D, allowing on-site degradation of TH and facilitating the intracellular release of siRNA. The NPs assembled with TH produced a higher silencing efficiency of target genes in vitro and in vivo than the NPs assembled with protamine as the nucleic acid condensing agent. The exploitation of TH in the NP formulation exhibited a biocompatibility profile similar to that of protamine, with minimal immunostimulating and systemic toxicity observed after repeated administration.     

Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.     

Silencing of Reporter Gene Expression in Skin Using siRNAs and Expression of Plasmid DNA Delivered by a Soluble Protrusion Array Device (PAD)

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.     

Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers

Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.     

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.     

Tumor vasculature is a key determinant for the efficiency of nanoparticle-mediated siRNA delivery

Delivering small interfering RNA (siRNA) to tumors using clinically viable formulations remains the primary technical hurdle that prevents the development of siRNA therapy for cancer treatment. Over the past several years, significant effort has been devoted to explore novel delivery strategies, whereas relatively little attention has been paid to understand the impact of physiological constrains such as tumor vasculature on the efficiency of siRNA delivery. Using the previously described positive-readout tumor models where successful siRNA delivery leads to an upregulation of β-galactosidase within tumor sections, we analyzed the spatial distribution of localized target knockdown within tumor sections relative to tumor hypoxia and found that stable nucleic acid lipid particle (SNALP), a lipid nanoparticle-based delivery system, predominantly delivers siRNA to areas adjacent to functional tumor blood vessels. Increasing tumor vascularity by ectopic expression of VEGF resulted in more efficient siRNA delivery to tumors using SNALP. SNALP-mediated delivery of a siRNA-targeting Ran GTPase led to target knockdown and significant antitumor efficacy in the highly vascularized HepG2-derived liver tumors, but not in the poorly vascularized HCT-116-derived liver tumors. These results highlight the significant impact of tumor vasculature on siRNA delivery and call for a more focused effort on addressing tumor penetration after extravasation, an area of only limited attention currently.     

A novel environment-sensitive biodegradable polydisulfide with protonatable pendants for nucleic acid delivery

Clinical application of nucleic acid-based therapies is limited by the lack of safe and efficient delivery systems. The purpose of this study is to design and evaluate novel biodegradable polymeric carriers sensitive to environmental changes for efficient delivery of nucleic acids, including plasmid DNA and siRNA. A novel polydisulfide with protonatable pendants was synthesized by the oxidative polymerization of a dithiol monomer, which was readily prepared by solid phase chemistry. The polydisulfide exhibited good buffering capacity and low cytotoxicity. It formed stable complexes with both plasmid DNA and siRNA. The particle sizes of the complexes decreased with the increase of the N/P ratios in the range of 100 to 750 nm. The complexes were stable in the presence of salt and heparin under normal physiological conditions, but dissociated to release nucleic acids in a reductive environment similar to cytoplasm. The polydisulfide demonstrated N/P ratio dependent transfection efficiency for plasmid DNA and gene silencing efficiency for siRNA. The presence of an endosomal disrupting agent, chloroquine, did not affect the DNA transfection efficiency of the polydisulfide. The transfection or gene silencing efficiency of the polydisulfide/DNA or siRNA complexes was comparable to or slightly lower than that of corresponding PEI complexes. Moreover, the polydisulfide showed better serum-friendly feature than PEI when delivering either DNA or siRNA in the presence of 10% FBS. This novel polydisulfide is a promising lead for further design and development of safe and efficient delivery systems for nucleic acids.     

Biocompatible gelatin nanoparticles for tumor-targeted delivery of polymerized siRNA in tumor-bearing mice

Structural modifications of the siRNA backbone improved its physiochemical properties for incorporating in gene carriers without loss of gene-silencing efficacy. These modifications provide a wider variety of choice of vector systems for siRNA delivery. We developed a tumor-targeted siRNA delivery system using polymerized siRNA (poly-siRNA) and natural polymer gelatin. The polymerized siRNA (poly-siRNA) was prepared through self-polymerization of thiol groups at the 5′-end of sense and anti-sense strands of siRNA and was encapsulated in the self-assembled thiolated gelatin (tGel) nanoparticles (NPs) with chemical cross-linking. The resulting poly-siRNA-tGel (psi-tGel) nanoparticles (average of 145 nm in diameter) protect siRNA molecules from enzymatic degradation, and can be reversibly reduced to release functional siRNA molecules in reductive conditions. The psi-tGel NPs presented efficient siRNA delivery in red fluorescence protein expressing melanoma cells (RFP/B16F10) to down-regulate target gene expression. In addition, the NPs showed low toxicity at a high transfection dose of 125 μg/ml psi-tGel NPs, which included 1 μM of siRNA molecules. In tumor-bearing mice, the psi-tGel NPs showed 2.8 times higher tumor accumulation than the naked poly-siRNA, suggesting tumor-targeted siRNA delivery of psi-tGel NPs. Importantly, the psi-tGel NPs induced effective tumor RFP gene silencing in vivo without remarkable toxicity. The psi-tGel NPs have great potential for a systemic siRNA delivery system for cancer therapy, based on their characteristics of low toxicity, tumor accumulation, and effective siRNA delivery.     

The Effect of Chemical Modification and Nanoparticle Formulation on Stability and Biodistribution of siRNA in Mice

Instability and inadequate biodistribution of double-stranded RNA are major drawbacks to the clinical use of RNA interference. This work compares chemical modification and nanoparticle formulation as strategies to improve the systemic delivery of small interfering RNA (siRNA). Variable levels of chemical modified siRNA, either naked or within nanoparticle, were intravenously injected into mice to study temporal stability and biodistribution detected by direct radioactive labeling or by northern blotting. Naked siRNA showed rapid renal clearance, with circulatory half-life of <5 minutes that could be extended to >30 minutes by cholesterol conjugation. The integrity of the chemically stabilized siRNA was maintained in blood for at least 30 minutes, whereas, unmodified siRNA duplex was degraded within 1 minute. Intact chemically modified siRNA could also be detected in all analyzed organs at 30 minutes but disappeared at 24 hours, except for heavy locked nucleic acid (LNA)-modified and cholesterol-conjugated siRNA in the lungs. Chitosan, liposomal, or JetPEI formulation greatly improved the stability and biodistribution of siRNA. Interestingly, high siRNA accumulation of the chitosan/siRNA formulation within the kidney was observed 24 hours postadministration. This comparative study highlights improvements to siRNA stability and pharmacokinetics, key determinants for development of clinically relevant RNAi therapeutics.     

siRNA nanocarriers based on methacrylic acid copolymers

Poly(ethylene glycol)-b-poly(propyl methacrylate-co-methacrylic acid) (PEG-b-P(PrMA-co-MAA) can be complexed with poly(amido amine) (PAMAM) dendrimers and nucleic acids to form pH-responsive nanosized core-shell type polyion complex micelles (PICMs). These PICMs have the ability to lose their shell and release the PAMAM/nucleic acid core under mildly acidic conditions such as those encountered in the endosomal compartment. In this work, pH-sensitive PICMs composed of PEG-b-P(PrMA-co-MAA), different PAMAMs, and siRNAs were prepared and characterized. These micelles had mean diameters ranging from 50 to 100 nm depending on the structure of the polycationic component. In order to trigger PICM uptake by receptor-mediated endocytosis, the micelles were decorated with an antibody fragment directed against the transferrin receptor (anti-CD71). The targeting ligand was stably conjugated to a semi-telechelic amino-PEG-b-P(PrMA-co-MAA) via a maleimide/activated ester bifunctional linker, yielding up to 60%-80% functionalization of the maleimide groups. The cellular uptake of the micelles was assessed on human prostate cancer cells (PC-3) via flow cytometry. Native PICMs and micelles bearing a non-specific antibody fragment were taken up to the same extent with a low efficiency, whereas anti-CD71 Fab′-decorated PICMs exhibited significantly higher uptake. The capacity of the targeted, siRNA-loaded, PICMs to downregulate the expression of the Bcl-2 anti-apoptotic oncoprotein was investigated using the appropriate unmodified or 2′-modified (2′F-RNA and 2′F-ANA) siRNA sequence. Bcl-2 mRNA and protein levels were greatly reduced when the cells were transfected with anti-CD71 decorated PICMs. Optimal silencing was achieved with the chemically modified siRNA. These data suggest that combining optimized siRNA chemistry with an effective delivery system can potentiate the activity of siRNA, thereby potentially reducing the total dose of carrier required to achieve a pharmacological effect.     

Transfection of human platelets with short interfering RNA

Platelets contain mRNAs and are capable of translating mRNA into protein, and it has been previously demonstrated that platelets increase their levels of integrin β3 overtime while in blood bank storage conditions. We are unaware of prior attempts to introduce nucleic acids into platelets. Considering the potential clinical and research utility of manipulating platelet gene expression, we tested whether small interfering RNAs (siRNAs) could be transfected into normal human platelets. Multiple conditions were tested, including lipofectamine versus electroporation, different amounts of siRNA, the effect of different buffers and the presence of plasma during transfection, and the time for optimal siRNA incorporation after transfection. Using flow cytometry to assess transfection efficiency, we found that optimal transfection was obtained using lipofectamine, washed platelets, and 400 pmoles siRNA. Cell sorting of transfected platelets suggested that the incorporated siRNA was able to knockdown the level of a targeted mRNA. This is the first ever demonstration that nucleic acids can be introduced directly into platelets, and offers proof of concept for manipulating gene expression in platelets by nonviral methods. Future technical improvements may permit improving the quality and/or lifespan of stored human platelets.     

An Amino Acid-based Amphoteric Liposomal Delivery System for Systemic Administration of siRNA

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA² compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA²-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA², cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA²-based liposomes demonstrate a linear dose–response with an ED₅₀ of 0.1 mg/kg against liver-specific target genes in BALB/c mice.     

Sonoporation as an Approach for siRNA delivery into T cells

Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm² for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future.     

In vivo silencing of a molecular target by short interfering RNA electroporation: tumor vascularization correlates to delivery efficiency

Screening for a molecular target for cancer therapy requires multiple steps, of which an important one is evaluation of the knockdown effect of the target molecule on pregrown xenograft tumors. However, methods currently used for local administration of knockdown reagents, such as short interfering RNA (siRNA), are not satisfactory as to simplicity and efficiency. We established an electroporation method involving a constant voltage and "plate and fork" type electrodes and used it for in vivo delivery of siRNA. The delivery efficiency correlated to the electric current. The electric current correlated to the microvascular density and vascular endothelial growth factor (VEGF) expression and exhibited a threshold that guaranteed efficient delivery. Consequently, we showed that the vascularization and VEGF expression in tumors determined the efficiency of delivery of siRNA by electroporation. VEGF was chosen as a model target. VEGF siRNA electroporation suppressed the growth of tumors exhibiting high VEGF expression to less than 10% of the control level, but it had no effect on low VEGF-expressing tumors. Notably, a long interval (20 days) of electroporation was enough to obtain a satisfactory effect. Systemically injected siRNA could also be delivered into tumors by this method. Our data will provide the technical basis for in vivo electroporation, and this simple and efficient siRNA delivery method is applicable to in vivo comprehensive screening for a molecular target.