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1.  Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells 
Molecular Therapy  2011;19(12):2186-2200.
Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.
PMCID: PMC3242662  PMID: 21971424
2.  Nanometer-scale siRNA carriers incorporating peptidomimetic oligomers: physical characterization and biological activity 
Synthetic short interfering RNA (siRNA) oligonucleotides can trigger the RNA interference pathway and lead to selective gene silencing. Despite considerable enthusiasm and investment, formidable challenges remain that may deter translating this breakthrough discovery into clinical applications. In particular, the development of efficient, nontoxic, nonimmunogenic methods for delivering siRNA in vivo has proven to be exceptionally challenging. Thorough analysis of the relationship between the structure and function of siRNA carrier systems, both in isolation and in complex with RNA, will facilitate the design of efficient nonviral siRNA delivery vehicles. In this study, we explore the relationship between the physicochemical characteristics and the biological activity of “lipitoid” compounds as potent siRNA delivery vehicles. Lipitoids are cationic peptidomimetic oligomers incorporating a peptoid and a phospholipid moiety. Lipitoids can associate with siRNA oligonucleotides and self-assemble into spherical lipitoid-based nanoparticles (LNPs), with dimensions that are dependent upon the medium and the stoichiometric ratio between the cationic monomers of the lipitoid and anionic siRNA oligonucleotides. The morphology, gene silencing efficiency, and cytotoxicity of the siRNA-loaded LNPs are similarly sensitive to the stoichiometry of the complexes. The medium in which the LNPs are formed affects the assembled cargo particles’ characteristics such as particle size, transfection efficiency, and stability. Formation of the LNPs in the biological, serum-free medium OptiMEM resulted in LNPs an order of magnitude larger than LNPs formed in water, and were twice as efficient in siRNA transfection compared to LNPs formed in water. Inhibitor studies were conducted to elucidate the efficiency of lysosomal escape and the uptake mechanism of the siRNA-loaded LNPs. Our results suggest that these lipitoid-based, siRNA-loaded spherical LNPs are internalized through a lipid raft-dependent and dynamin-mediated pathway, circumventing endosomal and lysosomal encapsulation. The lipitoid-siRNA nanospheres proved to be suitable platforms for investigating the critical parameters determining the efficiency of transfection agents, revealing the necessity for conducting characterization studies in biological media. The investigation of the LNP internalization pathway points to an alternative uptake route that bypasses the lysosome, explaining the surprisingly high efficiency of LNPs and suggesting that the uptake mechanism should be probed rather than assumed for the next generation of rationally designed transfection agents.
PMCID: PMC4026564  PMID: 24872690
lipitoid; siRNA delivery; therapeutic oligonucleotides; peptoid
3.  Lipidoid Nanoparticles for siRNA Delivery to the Intestinal Epithelium: In Vitro Investigations in a Caco-2 Model 
PLoS ONE  2015;10(7):e0133154.
Short interfering ribonucleic acid (siRNA) therapeutics show promise for the treatment of intestinal diseases by specifically suppressing the expression of disease relevant proteins. Recently, a class of lipid-like materials termed “lipidoids” have been shown to potently deliver siRNA to the liver and immune cells. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of siRNA delivery to the intestinal epithelium. Initial studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal epithelial cells, with a single 10 nM dose depressing GAPDH mRNA expression for one week. Transfection with siRNA-loaded LNPs did not induce significant cytotoxicity in Caco-2 cells or alter intestinal barrier function. Protein silencing was confirmed by Western blotting, with the lowest levels of GAPDH protein expression observed five days post-transfection. Together, these data underscore the potential of LNPs for the treatment of intestinal disorders.
PMCID: PMC4508104  PMID: 26192592
4.  Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide 
PLoS Genetics  2006;2(9):e140.
Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.
First discovered in nematodes, RNA interference (RNAi) has become an essential tool in the study of mammalian gene function. RNAi directed by small interfering RNAs (siRNAs), 21 nt, double-stranded RNAs target complementary mRNAs for destruction. siRNAs can be introduced into mammalian cells grown in culture, or even administered intravenously to rodents or primates, where they repress production of the targeted gene product. Thus, siRNA-directed RNAi has tremendous potential as a human therapeutic strategy. Dominant genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, might be treated with therapeutic siRNAs, provided that the siRNAs could be designed to destroy the mutant, disease-causing mRNA, while leaving the normal mRNA intact. Here, Schwarz and colleagues describe an experimentally validated strategy for the design of such siRNAs. Their design strategy should facilitate the design of siRNAs targeting dominant genetic disorders such as amyotrophic lateral sclerosis and Huntington disease.
PMCID: PMC1560399  PMID: 16965178
5.  A Myristoylated Cell-Penetrating Peptide Bearing a Transferrin Receptor-Targeting Sequence for Neuro-Targeted siRNA Delivery 
Molecular Pharmaceutics  2014;11(2):486-495.
Many neurodegenerative disorders (NDDs) are characterized by aggregation of aberrant proteins and extensive oxidative stress in brain cells. As a treatment option for NDDs, RNA interference (RNAi) is a promising approach to suppress the activation of abnormal genes and negative regulators of antioxidant genes. Efficient neuro-targeted siRNA delivery requires a delicate optimization of nucleic acid carriers, quite distinct from putative pDNA carriers in regard to stable condensation and serum protection of siRNA, blood–brain barrier (BBB) bypass, effective siRNA delivery to brain cells, and functional release of bioactive siRNA at therapeutic levels. Here, we propose that a myristic acid conjugated, cell-penetrating peptide (transportan; TP), equipped with a transferrin receptor-targeting peptide (myr-TP-Tf), will lead to stable encapsulation of siRNA and targeted delivery of siRNA to brain cells overcoming the BBB. Myr-TP-Tf was successfully prepared by solid-phase peptide synthesis with high purity. Myr-TP-Tf–siRNA complexes formulated at a 20:1 (peptide–siRNA) molar ratio provided prolonged siRNA stability against serum and ribonuclease treatment. Fluorescence images clearly indicated that siRNA uptake was successfully achieved by myr-TP-Tf complexes in both a murine brain endothelioma and a human glioma cell line. The luciferase assay and the human placental alkaline phosphatase (hPAP) reporter assay results demonstrated the functional gene silencing effect of myr-TP-Tf–siRNA complexes in a human glioma cell line as well as in primary murine neurons/astrocytes, supportive of successful release of bioactive siRNA into the cytosol. Finally, the transcytosis assay revealed that favorable siRNA transport via receptor-mediated transcytosis was mediated by myr-TP-Tf complexes. In summary, these data suggest that myr-TP-Tf peptides possess promising properties as a vehicle for neuro-targeted siRNA delivery. We will further study this peptide in vitro and in vivo for transport mechanism kinetics and to validate its capability to deliver siRNA to the brain, respectively.
PMCID: PMC3993914  PMID: 24387132
siRNA carrier; cell-penetrating peptide; blood−brain barrier (BBB); transferrin receptor; receptor-mediated transcytosis; neuro-targeting; neurodegenerative disorders (NDDs)
6.  Fusogenic-Oligoarginine Peptide-Mediated Delivery of siRNAs Targeting the CIP2A Oncogene into Oral Cancer Cells 
PLoS ONE  2013;8(9):e73348.
Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.
PMCID: PMC3760901  PMID: 24019920
7.  Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA 
Science translational medicine  2014;6(250):250ra116.
Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)–targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti–MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.
PMCID: PMC4502585  PMID: 25143366
8.  siRNA applications in nanomedicine 
The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease or the augmentation of tissue formation. RNAi is the sequence-specific gene silencing mediated by a 21 to 25 nucleotide double stranded small interfering RNA (siRNA) molecule. siRNAs are incorporated into the RNAi-induced silencing complex (RISC), which mediates mRNA sequence specific binding and cleavage. Although RNAi has the potential to be a powerful therapeutic drug, its delivery remains a major limitation. The generation of nano-sized particles is being investigated to enhance the delivery of siRNA-based drugs. These nanoparticles are generally designed to overcome one or more of the barriers encountered by the siRNA when trafficked to the cytosol. In this review, we will discuss recent advances in the design of delivery strategies for siRNA, focusing our attention to those strategies that have had in vivo success or have introduced novel functionality that allowed enhanced intracellular trafficking and/or cellular targeting. The review will first discuss the different barriers that must be overcome for efficient siRNA delivery. Second we will discuss the approaches for siRNA delivery by size including direct modification of siRNAs (less than 10nm), self-assembled particles based on cationic polymers and cationic lipids (100 to 300 nm), neutral liposomes (< 200 nm), and macro scale matrices that contain naked siRNA or siRNA loaded nanoparticles (> 100 μm). Last, we will briefly discuss recent in vivo therapeutic successes.
PMCID: PMC4104279  PMID: 20135697
siRNA; Non-viral gene delivery; RNAi; siRNA nanoparticles; Nanomedicine
9.  TLR agonist–Stat3 siRNA conjugates: cell-specific gene silencing and enhanced antitumor immune responses 
Nature biotechnology  2009;27(10):925-932.
Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.
PMCID: PMC2846721  PMID: 19749770
10.  Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA 
Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.
PMCID: PMC3442367  PMID: 23344179
lipid nanoparticle; microfluidics; nanomedicine; siRNA; synthesis and formulation
11.  Versatile RNA Interference Nanoplatform for Systemic Delivery of RNAs 
ACS Nano  2014;8(5):4559-4570.
Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells.
PMCID: PMC4046792  PMID: 24779637
RNAi; nanomedicine; gene and drug delivery; hyaluronic acid; cancer therapy
12.  Lipid Nanoparticles for Hepatic Delivery of Small Interfering RNA 
Biomaterials  2012;33(25):5924-5934.
Clinical application of small interfering RNA (siRNA) requires safe and efficient delivery in vivo. Here, we report the design and synthesis of lipid nanoparticles (LNPs) for siRNA delivery based on cationic lipids with multiple tertiary amines and hydrophobic linoleyl chains. LNPs incorporating the lipid containing tris(2-aminoethyl)amine (TREN) and 3 linoleyl chain, termed TRENL3, were found to have exceptionally high siRNA transfection efficacy that was markedly superior to lipofectamine, a commercial transfection agent. In addition, inclusion of polyunsaturated fatty acids, such as linoleic acid and linolenic acids in the formulation further enhanced the siRNA delivery efficiency. TRENL3 LNPs were further shown to transported siRNA into the cytosol primarily via macropinocytosis rather than clathrin-mediated endocytosis. The new LNPs have demonstrated preferential uptake by the liver and hepatocellular carcinoma in mice, thereby leading to high siRNA gene silencing activity. These data suggest potential therapeutic applications of TRENL3 mediated delivery of siRNA for liver diseases.
PMCID: PMC3374058  PMID: 22652024
Cationic lipids; Lipid nanoparticles; Small interfering RNA; hepatocellular carcinoma
13.  Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics 
Theranostics  2014;4(12):1211-1232.
Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA.
PMCID: PMC4183999  PMID: 25285170
gene delivery; gene silencing; nanoparticles; non-viral vectors; small interfering RNA (siRNA).
14.  Exosomes are natural carriers of exogenous siRNA to human cells in vitro 
Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies.
This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells.
The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy.
PMCID: PMC3895799  PMID: 24245560
Exosomes; RNA interference (RNAi); Drug delivery system; Cancer therapy; RAD51
15.  Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo 
RNA interference is an evolutionary conserved immune response mechanism that can be used as a tool to provide novel insights into gene function and structure. The ability to efficiently deliver small interfering RNA to modulate gene expression in vivo may provide new therapeutic approaches to currently intractable diseases.
In vitro, siRNA targeting IL-12p40 was delivered to the murine macrophage cell line (J774A.1) encapsulated in a liposome with an IL-12 inducing agent (LPS/IFN-γ) over a number of time points. Controls included a variety of non-target specific siRNA reagents. Supernatants were analyzed for cytokine production while the cells were removed for mRNA profiling.
In vivo, siRNA-targeting IL-12p40 was delivered to the murine peritoneal cavity in a therapeutic fashion, after endotoxin (LPS) challenge. Cells from the peritoneal cavity were removed by lavage and analyzed by flow cytometry. Levels of IL-12 present in lavage and in serum were also examined by ELISA.
In this report, we show that IL-12p40 siRNA can specifically silence macrophage expression of IL-12p40 mRNA and IL-12p70 protein in vitro. We extend this finding to demonstrate that delivery of liposome encapsulated siRNA targeting IL-12p40 to the murine peritoneal cavity can modulate an inflammatory stimulus in vivo. Furthermore, specific siRNA can be used therapeutically after endotoxin challenge to reduce both the local and systemic inflammatory response. Thus, the delivery of siRNA can be used to elicit specific non-permanent inhibition of endogenous protein expression.
In vitro silencing of IL-12p40 using siRNA at selected doses leads to specific knockdown of IL-12p70 protein production without inducing type I interferons. Furthermore, siRNA targeting murine IL-12p40 can be used therapeutically to counter an inflammatory response in vivo.
PMCID: PMC1074346  PMID: 15813981
16.  mRNA turnover rate limits siRNA and microRNA efficacy 
Based on a simple model of the mRNA life cycle, we predict that mRNAs with high turnover rates in the cell are more difficult to perturb with RNAi.We test this hypothesis using a luciferase reporter system and obtain additional evidence from a variety of large-scale data sets, including microRNA overexpression experiments and RT–qPCR-based efficacy measurements for thousands of siRNAs.Our results suggest that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
What determines how strongly an mRNA responds to a microRNA or an siRNA? We know that properties of the sequence match between the small RNA and the mRNA are crucial. However, large-scale validations of siRNA efficacies have shown that certain transcripts remain recalcitrant to perturbation even after repeated redesign of the siRNA (Krueger et al, 2007). Weak response to RNAi may thus be an inherent property of the mRNA, but the underlying factors have proven difficult to uncover.
siRNAs induce degradation by sequence-specific cleavage of their target mRNAs (Elbashir et al, 2001). MicroRNAs, too, induce mRNA degradation, and ∼80% of their effect on protein levels can be explained by changes in transcript abundance (Hendrickson et al, 2009; Guo et al, 2010). Given that multiple factors act simultaneously to degrade individual mRNAs, we here consider whether variable responses to micro/siRNA regulation may, in part, be explained simply by the basic dynamics of mRNA turnover. If a transcript is already under strong destabilizing regulation, it is theoretically possible that the relative change in abundance after the addition of a novel degrading factor would be less pronounced compared with a stable transcript (Figure 1). mRNA turnover is achieved by a multitude of factors, and the influence of such factors on targetability can be explored. However, their combined action, including yet unknown factors, is summarized into a single property: the mRNA decay rate.
First, we explored the theoretical relationship between the pre-existing turnover rate of an mRNA, and its expected susceptibility to perturbation by a small RNA. We assumed a basic model of the mRNA life cycle, in which the rate of transcription is constant and the rate of degradation is described by first-order kinetics. Under this model, the relative change in steady-state expression level will become smaller as the pre-existing decay rate grows larger, independent of the transcription rate. This relationship persists also if we assume various degrees of synergy and antagonism between the pre-existing factors and the external factor, with increasing synergism leading to transcripts being more equally targetable, regardless of their pre-existing decay rate.
We next generated a series of four luciferase reporter constructs with destabilizing AU-rich elements (AREs) of various strengths incorporated into their 3′ UTRs. To evaluate how the different constructs would respond to perturbation, we performed co-transfections with an siRNA targeted at the coding region of the luciferase gene. This reduced the signal of the non-destabilized construct to 26% compared with a control siRNA. In contrast, the most destabilized construct showed 42% remaining reporter activity, and we could observe a dose–response relationship across the series.
The reporter experiment encouraged an investigation of this effect on real-world mRNAs. We analyzed a set of 2622 siRNAs, for which individual efficacies were determined using RT–qPCR 48 h post-transfection in HeLa cells ( Of these, 1778 could be associated with an experimentally determined decay rate (Figure 4A). Although the overall correlation between the two variables was modest (Spearman's rank correlation rs=0.22, P<1e−20), we found that siRNAs directed at high-turnover (t1/2<200 min) and medium-turnover (2001000 min) transcripts (P<8e−11 and 4e−9, respectively, two-tailed KS-test, Figure 4B). While 41.6% (498/1196) of the siRNAs directed at low-turnover transcripts reached 10% remaining expression or better, only 16.7% (31/186) of the siRNAs that targeted high-turnover mRNAs reached this high degree of silencing (Figure 4B). Reduced targetability (25.2%, 100/396) was also seen for transcripts with medium-turnover rate.
Our results based on siRNA data suggested that turnover rates could also influence microRNA targeting. By assembling genome-wide mRNA expression data from 20 published microRNA transfections in HeLa cells, we found that predicted target mRNAs with short and medium half-life were significantly less repressed after transfection than their long-lived counterparts (P<8e−5 and P<0.03, respectively, two-tailed KS-test). Specifically, 10.2% (293/2874) of long-lived targets versus 4.4% (41/942) of short-lived targets were strongly (z-score <−3) repressed. siRNAs are known to cause off-target effects that are mediated, in part, by microRNA-like seed complementarity (Jackson et al, 2006). We analyzed changes in transcript levels after transfection of seven different siRNAs, each with a unique seed region (Jackson et al, 2006). Putative ‘off-targets' were identified by mapping of non-conserved seed matches in 3′ UTRs. We found that low-turnover mRNAs (t1/2 >1000 min) were more affected by seed-mediated off-target silencing than high-turnover mRNAs (t1/2 <200 min), with twice as many long-lived seed-containing transcripts (3.8 versus 1.9%) being strongly (z-score <−3) repressed.
In summary, mRNA turnover rates have an important influence on the changes exerted by small RNAs on mRNA levels. It can be assumed that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
The microRNA pathway participates in basic cellular processes and its discovery has enabled the development of si/shRNAs as powerful investigational tools and potential therapeutics. Based on a simple kinetic model of the mRNA life cycle, we hypothesized that mRNAs with high turnover rates may be more resistant to RNAi-mediated silencing. The results of a simple reporter experiment strongly supported this hypothesis. We followed this with a genome-wide scale analysis of a rich corpus of experiments, including RT–qPCR validation data for thousands of siRNAs, siRNA/microRNA overexpression data and mRNA stability data. We find that short-lived transcripts are less affected by microRNA overexpression, suggesting that microRNA target prediction would be improved if mRNA turnover rates were considered. Similarly, short-lived transcripts are more difficult to silence using siRNAs, and our results may explain why certain transcripts are inherently recalcitrant to perturbation by small RNAs.
PMCID: PMC3010119  PMID: 21081925
microRNA; mRNA decay; RNAi; siRNA
17.  A Novel In Vivo siRNA Delivery System Specifically Targeting Liver Cells for Protection of ConA-Induced Fulminant Hepatitis 
PLoS ONE  2012;7(9):e44138.
Fulminant hepatitis progresses to acute liver failure (ALF) when the extent of hepatocyte death exceeds the liver's regenerative capacity. Although small interfering RNA (siRNA) appears promising in animal models of hepatitis, the approach is limited by drawbacks associated with systemic administration of siRNA. The aim of this study is to develop a hepatocyte-specific delivery system of siRNA for treatment of fulminant hepatitis.
Methodology/Principal Findings
Galactose-conjugated liposome nano-particles (Gal-LipoNP) bearing siRNA was prepared, and the particle size and zeta potential of Gal-LipoNP/siRNA complexes were measured. The distribution, cytotoxicity and gene silence efficiency were studied in vivo in a concanavalin A (ConA)-induced hepatitis model. C57BL/6 mice were treated with Gal-LipoNP Fas siRNA by i.v. injection 72 h before ConA challenge, and hepatocyte injury was evaluated using serum alanine transferase (ALT) and aspartate transaminase (AST) levels, as well as liver histopathology and TUNEL-positive hepatocytes. The galactose-ligated liposomes were capable of encapsulating >96% siRNA and exhibited a higher stability than naked siRNA in plasma. Hepatocyte-specific targeting was confirmed by in vivo delivery experiment, in which the majority of Gal-LipoNP-siRNA evaded nuclease digestion and accumulated in the liver as soon as 6 h after administration. In vivo gene silencing was significant in the liver after treatment of Gal-Lipo-siRNA. In the ConA-induced hepatitis model, serum levels of ALT and AST were significantly reduced in mice treated with Gal-lipoNP-siRNA as compared with control mice. Additionally, tissue histopathology and apoptosis showed an overall reduction of injury in the Gal-LipoNP siRNA-treated mice.
This study is the first to our knowledge to demonstrate reduction of hepatic injury by liver-specific induction of RNA interference using Gal-LipoNP Fas siRNA, highlighting a novel RNAi-based therapeutic potential in many liver diseases.
PMCID: PMC3435394  PMID: 22970170
18.  RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes 
T cells play a central role in many inflammatory diseases, hence the identification and validation of T cell-specific target genes will increase the understanding of T cell function in pathologic inflammatory situations. RNA interference (RNAi), with its ability to induce specific gene silencing in mammalian cells, represents a powerful technology to investigate and validate the function of pharmaceutical target genes in vitro and in vivo. The aim of the present study was to systematically explore RNAi-mediated gene-silencing of known T cell-specific model signaling molecules in primary murine T cells in vitro and in vivo.
We demonstrate that siRNA delivery and subsequent silencing of T cell specific genes is substantially increased, if murine T cells were activated prior siRNA transfection. Silencing of ZAP70, p56Lck as well as PLC-γ1 protein expression resulted in impaired function of T cells in vitro. Furthermore, delayed type hypersensitivity (DTH) was ameliorated in vivo after adoptive transfer of ZAP70-silenced T cells.
The combination of RNAi-mediated gene silencing and adoptive transfer of gene-silenced T cells, thus, may allow the identification and analysis of T cell-specific targets for therapeutic intervention. Additionally, this model system may represent an alternative to conventional time consuming and cost intensive gene targeting approaches.
PMCID: PMC2517589  PMID: 18684324
19.  Investigation of enzyme-sensitive lipid nanoparticles for delivery of siRNA to blood–brain barrier and glioma cells 
Clinical applications of siRNA for treating disorders in the central nervous system require development of systemic stable, safe, and effective delivery vehicles that are able to cross the impermeable blood–brain barrier (BBB). Engineering nanocarriers with low cellular interaction during systemic circulation, but with high uptake in targeted cells, is a great challenge and is further complicated by the BBB. As a first step in obtaining such a delivery system, this study aims at designing a lipid nanoparticle (LNP) able to efficiently encapsulate siRNA by a combination of titratable cationic lipids. The targeted delivery is obtained through the design of a two-stage system where the first step is conjugation of angiopep to the surface of the LNP for targeting the low-density lipoprotein receptor-related protein-1 expressed on the BBB. Second, the positively charged LNPs are masked with a negatively charged PEGylated (poly(ethylene glycol)) cleavable lipopeptide, which contains a recognition sequence for matrix metalloproteinases (MMPs), a class of enzymes often expressed in the tumor microenvironment and inflammatory BBB conditions. Proteolytic cleavage induces PEG release, including the release of four glutamic acid residues, providing a charge switch that triggers a shift of the LNP charge from weakly negative to positive, thus favoring cellular endocytosis and release of siRNA for high silencing efficiency. This work describes the development of this two-stage nanocarrier-system and evaluates the performance in brain endothelial and glioblastoma cells with respect to uptake and gene silencing efficiency. The ability of activation by MMP-triggered dePEGylation and charge shift is demonstrated to substantially increase the uptake and the silencing efficiency of the LNPs.
PMCID: PMC4590347  PMID: 26451106
matrix metalloproteinase; cleavable PEG-lipid; gene therapy; BBB; angiopep; nanocarrier
20.  Low-weight polyethylenimine cross-linked 2-hydroxypopyl-β-cyclodextrin and folic acid as an efficient and nontoxic siRNA carrier for gene silencing and tumor inhibition by VEGF siRNA 
Targeted delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. However, the need for safe and efficient delivery systems is a barrier to further development of RNA interference therapeutics. In this work, a nontoxic and efficient siRNA carrier delivery system of low molecular weight polyethyleneimine (PEI-600 Da) cross-linked with 2-hydroxypopyl-β-cyclodextrin (HP-β-CD) and folic acid (FA) was synthesized for biomedical application.
The siRNA carrier was prepared using a simple method and characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The siRNA carrier nanoparticles were characterized in terms of morphology, size and zeta potential, stability, efficiency of delivery, and gene silencing efficiency in vitro and in vivo.
The siRNA carrier was synthesized successfully. It showed good siRNA binding capacity and ability to protect siRNA. Further, the toxicity of the carrier measured in vitro and in vivo appeared to be negligible, probably because of degradation of the low molecular weight PEI and HP-β-CD in the cytosol. Flow cytometry and confocal microscopy confirmed that the FA receptor-mediated endocytosis of the FA-HP-β-CD-PEI/siRNA complexes was greater than that of the HP-β-CD-PEI/siRNA complexes in FA receptor-enriched HeLa cells. The FA-HP-β-CD-PEI/siRNA complexes also demonstrated excellent gene silencing efficiency in vitro (in the range of 90%), and reduced vascular endothelial growth factor (VEGF) protein expression in the presence of 20% serum. FA-HP-β-CD-PEI/siRNA complexes administered via tail vein injection resulted in marked inhibition of tumor growth and reduced VEGF protein expression in the tumors.
Our results suggest that the FA-HP-β-CD-PEI complex is a nontoxic and highly efficient gene carrier with the potential to deliver siRNA for cancer gene therapy effectively in vitro and in vivo.
PMCID: PMC3678862  PMID: 23766646
polyethyleneimine; 2-hydroxypropyl-β-cyclodextrin; folic acid; siRNA carrier; vascular endothelial growth factor; gene silencing
21.  Dual Functional RNA Nanoparticles Containing Phi29 Motor pRNA and Anti-gp120 Aptamer for Cell-type Specific Delivery and HIV-1 Inhibition 
Methods (San Diego, Calif.)  2011;54(2):284-294.
The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficient delivery of siRNAs remains a key obstacle to successful application. A targeted intracellular delivery approach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interactions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral membrane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at the cell surface. We previously described a novel dual inhibitory anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstrated that gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs.
Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing a HIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA packaging motor. We demonstrate that pRNA-aptamer chimeras specifically bind to and are internalized into cells expressing HIV gp120. Moreover, the pRNA-aptamer chimeras alone also provide HIV inhibitory function by blocking viral infectivity. The Ab′ pRNA-siRNA chimera with 2′-F modified pyrimidines in the sense strand not only improved the RNA stability in serum, but also was functionally processed by Dicer, resulting in specific target gene silencing. Therefore, this dual functional pRNA-aptamer not only represents a potential HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise for systemic anti-HIV therapy.
PMCID: PMC3107903  PMID: 21256218
RNAi; Anti-gp120 aptamer; nanobiotechnology; bionanotechnology; nanotechnology; AIDS Treatment; viral DNA packaging; nanomotors
22.  Tracking in vitro and in vivo siRNA electrotransfer in tumor cells 
RNA interference-mediated gene silencing offers the potential of targeted inhibition of disease-relevant genes. In vivo delivery of RNAi reagents can be obtained by a variety of approaches. Physical delivery methods appear safer and lack side effects. Electro-permeabilization is one of the non-viral methods successfully used to transfer small interfering RNAs (siRNAs) in vitro and in vivo. A promising approach may be, very little is known about the fundamental processes mediating siRNA transfer. In this study, we have investigated cellular delivery pathways involved in electro-delivery of siRNAs by a direct fluorescence imaging method. An Alexa-labeled siRNA was electro-transferred into murine melanoma cells stably-expressing the enhanced green fluorescent protein (eGFP) target reporter gene. The silencing of eGFP gene expression was quantified by time-lapsed fluorescence microscopy. Fluorescently-labeled siRNAs were found distributed homogeneously in cytoplasm 48 hours after electro-transfer, apparently by diffusion. Furthermore, siRNAs showed homogeneous distribution in vivo 48 hrs after intra-tumoral injection followed by electro- permeabilization. Histological fluorescence microscopy showed that siRNAs were mostly localized in the cytoplasm. Overall, this study shows that electro-permeabilization facilitates cytoplasmic distribution of siRNA, both in cultured cells and in vivo. This method offers a potential therapeutic tool to facilitate direct siRNA penetration into solid tumors.
PMCID: PMC2737239  PMID: 19771237
Electro-permeabilization; electro-poration; RNAi; tumors; fluorescence microscopy
23.  Targeting Stat3 in the myeloid compartment drastically improves the in vivo antitumor functions of adoptively transferred T cells 
Cancer research  2010;70(19):7455-7464.
Improving effector T cell functions is highly desirable for preventive or therapeutic interventions of diverse diseases. Stat3 in the myeloid compartment constrains Th-1 type immunity, dampening natural and induced antitumor immune responses. We have recently developed an in vivo siRNA delivery platform by conjugating a TLR9 agonist with siRNA that efficiently targets myeloid and B cells. Here we show that either ablating the Stat3 alleles in the myeloid compartment and B cells combined with CpG triggering or administrating the CpG-Stat3siRNA conjugates drastically augments effector functions of adoptively transferred CD8+ T cells. Specifically, we demonstrate that both approaches are capable of increasing dendritic cell and CD8+ T cell engagement in tumor draining lymph nodes. Furthermore, both approaches can significantly activate the transferred CD8+ T cells in vivo, upregulating effector molecules such as perforin, granzyme B and IFN-γ. Intravital multiphoton microscopy reveals that Stat3 silencing combined with CpG triggering greatly increases killing activity and tumor infiltration of transferred T cells. These results suggest the use of CpG-Stat3siRNA, and possibly other Stat3 inhibitors, as a potent adjuvant to improve T cell therapies.
PMCID: PMC3058618  PMID: 20841481
24.  Lipid nanoparticle siRNA treatment of Ebola virus Makona infected nonhuman primates 
Nature  2015;521(7552):362-365.
The current outbreak of Ebola virus (EBOV) in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled1. Several postexposure interventions have been employed under compassionate use to treat a number of patients repatriated to Europe and the United States2. However, the in vivo efficacy of these interventions against the new outbreak strain of EBOV is unknown. Here, we show that lipid nanoparticle (LNP)-encapsulated siRNAs rapidly adapted to target the Makona outbreak strain of EBOV are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days postexposure while animals were viremic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal hematology, blood chemistry, and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered while the untreated control animals succumbed. These results represent the first successful demonstration of therapeutic anti-EBOV efficacy against the new outbreak strain in nonhuman primates (NHPs) and highlight the rapid development of LNP-delivered siRNA as a countermeasure against this highly lethal human disease.
PMCID: PMC4467030  PMID: 25901685
25.  Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles 
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.
PMCID: PMC3894582  PMID: 24345865
drug delivery; hepatocyte; lipid nanoparticles; polyethylene glycol; siRNA; prenatal diagnosis

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