Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma.

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway.

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (approximately 80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.

Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Modulation of thermal stability can enhance the potency of siRNA.

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as non-canonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9-12 nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potency-enhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

Characterization of side reactions during the annealing of small interfering RNAs.

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions-such as 2',3'-isomerization, strand scission, and HF elimination-depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.

Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells.

Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.

Polyethylenimine/small interfering RNA-mediated knockdown of vascular endothelial growth factor in vivo exerts anti-tumor effects synergistically with Bevacizumab.

BACKGROUND: RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA-based therapies and, recently, the novel branched PEI F25-LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy. METHODS: In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25-LMW/siRNA-mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti-tumorigenic efficacies of: (i) PEI/siRNA-mediated VEGF targeting; (ii) treatment with the monoclonal anti-VEGF antibody Bevacizumab (Avastin); and (iii) a combination of both. RESULTS: Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side-effects of PEI F25-LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti-tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed. CONCLUSIONS: PEI F25-LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti-tumor therapy, and PEI/siRNA-mediated, therapeutic VEGF knockdown exerts anti-tumor effects.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy.

We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

FAPP2 gene downregulation increases tumor cell sensitivity to Fas-induced apoptosis.

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods.

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.

DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization.

siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4-10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3' end of the siRNA passenger strand was beneficial over the 5' end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol-shielded polyplex to downregulate a luciferase gene in folate receptor-positive cells.

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome.

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.

Small interfering RNA targeting bcl-2 sensitizes malignant melanoma.

Malignant melanoma is a prime example of a treatment-resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low, and the duration of responses is short. More than 90% of all melanomas express the antiapoptotic protein Bcl-2, shown to contribute to a chemoresistant phenotype in melanoma. We previously demonstrated that antisense-mediated inhibition of Bcl-2 sensitizes malignant melanoma to apoptosis-inducing treatment modalities. In the present study, we evaluated synthetic small interfering RNA (siRNA) compounds targeting Bcl-2 as a novel approach to downregulate Bcl-2 expression in melanoma cells. siRNA treatment led up to a 19-fold reduction of bcl-2 mRNA levels and only barely detectable Bcl-2 protein expression at low nanomolar concentrations. Silencing of Bcl-2 in melanoma cells by specific siRNA led to a moderate increase in apoptotic cell death and inhibition of cell growth. However, if siRNA compounds targeting Bcl-2 were combined with the apoptosis-inducing chemotherapeutic agent cisplatin, a massive increase in apoptotic cell death compared with controls was observed. Notably, the combination of Bcl2 siRNA and low-dose cisplatin resulted in a supra-additive effect, with nearly complete suppression of cell growth, whereas cell growth in cisplatin-only-treated cells was only moderately affected (96% vs. 25%, p < 0.001). These findings underline a key role for Bcl-2 in conferring chemoresistance to melanoma and highlight Bcl-2 siRNA strategies as novel and highly effective tools, with the potential for future targeted therapy of malignant melanoma.

Defined Folate-PEG-siRNA Conjugates for Receptor-specific Gene Silencing.

Gene silencing mediated by small interfering RNA (siRNA) is a novel approach in the development of new cancer therapeutics. Polycations used for nucleic acid delivery still remain heterogeneous compounds, despite continuous progress in polymer synthetic technologies. Here we report the development of a structural defined folic acid polyethylene glycol (PEG) siRNA conjugate accessible via click chemistry yielding a monodisperse ligand-PEG-siRNA conjugate. The folic acid targeting ligand was synthesized by solid phase supported peptide chemistry. The conjugate was shown to be specifically internalized into folic acid receptor expressing cells. When combined with a structurally defined polycation, again synthesized with the precision of solid phase chemistry, efficient receptor specific gene silencing is achieved.