Knockdown Of Target Genes Research Articles

Modeling time-dependent transcription effects of HER2 oncogene and discovery of a role for E2F2 in breast cancer cell-matrix adhesion.

Oncogenes are known drivers of cancer phenotypes and targets of molecular therapies; however, the complex and diverse signaling mechanisms regulated by oncogenes and potential routes to targeted therapy resistance remain to be fully understood. To this end, we present an approach to infer regulatory mechanisms downstream of the HER2 driver oncogene in SUM-225 metastatic breast cancer cells from dynamic gene expression patterns using a succession of analytical techniques, including a novel MP grammars method to mathematically model putative regulatory interactions among sets of clustered genes. Our method highlighted regulatory interactions previously identified in the cell line and a novel finding that the HER2 oncogene, as opposed to the proto-oncogene, upregulates expression of the E2F2 transcription factor. By targeted gene knockdown we show the significance of this, demonstrating that cancer cell-matrix adhesion and outgrowth were markedly inhibited when E2F2 levels were reduced. Thus, validating in this context that upregulation of E2F2 represents a key intermediate event in a HER2 oncogene-directed gene expression-based signaling circuit. This work demonstrates how predictive modeling of longitudinal gene expression data combined with multiple systems-level analyses can be used to accurately predict downstream signaling pathways. Here, our integrated method was applied to reveal insights as to how the HER2 oncogene drives a specific cancer cell phenotype, but it is adaptable to investigate other oncogenes and model systems. Accessibility of various tools is listed in methods; the Log-Gain Stoichiometric Stepwise algorithm is accessible at http://www.cbmc.it/software/Software.php.

Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BackgroundThe dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.ResultsIn order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.ConclusionThe unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation.

BackgroundTamoxifen resistance is a major problem in the treatment of estrogen receptor (ER) α -positive breast cancer patients. Although the mechanisms behind tamoxifen resistance are still not completely understood, clinical data suggests that increased expression of receptor tyrosine kinases is involved. Here, we studied the estrogen and anti-estrogen sensitivity of human breast cancer MCF7 cells that have a moderate, retroviral-mediated, ectopic expression of epidermal growth factor receptor (MCF7-EGFR).MethodsProliferation of MCF7-EGFR and parental cells was induced by 17β-estradiol (E2), epidermal growth factor (EGF) or a combination of these. Inhibition of proliferation under these conditions was investigated with 4-hydroxy-tamoxifen (TAM) or fulvestrant at 10-12 to 10-6 M. Cells were lysed at different time points to determine the phosphorylation status of EGFR, MAPK1/3, AKT and the expression of ERα. Knockdown of target genes was established using smartpool siRNAs. Transcriptomics analysis was done 6 hr after stimulation with growth factors using Affymetrix HG-U133 PM array plates.ResultsWhile proliferation of parental MCF7 cells could only be induced by E2, proliferation of MCF7-EGFR cells could be induced by either E2 or EGF. Treatment with TAM or fulvestrant did significantly inhibit proliferation of MCF7-EGFR cells stimulated with E2 alone. EGF treatment of E2/TAM treated cells led to a marked cell proliferation thereby overruling the anti-estrogen-mediated inhibition of cell proliferation. Under these conditions, TAM however did still inhibit ERα- mediated transcription. While siRNA-mediated knock-down of EGFR inhibited the EGF- driven proliferation under TAM/E2/EGF condition, knock down of ERα did not. The TAM resistant cell proliferation mediated by the conditional EGFR-signaling may be dependent on the PI3K/Akt pathway but not the MEK/MAPK pathway, since a MEK inhibitor (U0126), did not block the proliferation. Transcriptomic analysis under the various E2/TAM/EGF conditions revealed that E2 and EGF dependent transcription have little overlap and rather operate in a parallel fashion.ConclusionsOur data indicate that enhanced EGFR-driven signalling is sufficient to overrule the TAM- mediated inhibition of E2-driven cell proliferation. This may have profound implications for the anti-estrogen treatment of ER-positive breast cancers that have increased levels of EGFR.

A method to assess target gene involvement in angiogenesis in vitro and in vivo using lentiviral vectors expressing shRNA.

Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was signifcantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.

Cationic lipid nanodisks as an siRNA delivery vehicle.

The term nanodisk (ND) describes reconstituted high-density lipoprotein particles that contain one or more exogenous bioactive agents. In the present study, ND were assembled from apolipoprotein A-I, the zwitterionic glycerophospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the synthetic cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). ND formulated at a DMPC:DMTAP ratio of 70:30 (by weight) were soluble in aqueous media. The particles generated were polydisperse, with diameters ranging from ∼20 to <50 nm. In nucleic acid binding studies, agarose gel retardation assays revealed that a synthetic 23-mer double-stranded oligonucleotide (dsOligo) bound to DMTAP containing ND but not to ND formulated with DMPC alone. Sucrose density gradient ultracentrifugation studies provided additional evidence for stable dsOligo binding to DMTAP-ND. Incubation of cultured hepatoma cells with DMTAP-ND complexed with a siRNA directed against glyceraldehyde 3-phosphate dehydrogenase showed 60% knockdown efficiency. Thus, incorporation of synthetic cationic lipid (i.e., DMTAP) to ND confers an ability to bind siRNA and the resulting complexes possess target gene knockdown activity in a cultured cell model.

Delivery of EZH2-shRNA with mPEG-PEI nanoparticles for the treatment of prostate cancer in vitro

Small interfering RNA (siRNA) is a promising therapeutic approach for castration-resistant prostate cancer (PCa). For the clinical application of siRNA, it is vital to find a safe and efficient gene transfer vector. Nanotechnology can provide a crucial advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of new therapeutic delivery vehicles. In this study, we describe a novel nanoparticle (mPEG-PEI) as an efficient non-viral carrier and found that this copolymer displayed enhanced efficiency in the shRNA-mediated knockdown of target genes. The enhancer of zeste homolog 2 (EZH2) is often elevated in castration-resistant PCa and has been implicated in the progression of human PCa. Targeting EZH2 may have therapeutic efficacy for the treatment of metastatic, hormone-refractory PCa. mPEG-PEI binds plasmid DNA yielding nanoparticles and these complexes exhibit low cytotoxicity and high gene transfection efficiency. Taken together, mPEG-PEI may be a promising non-viral gene carrier for the delivery of EZH2 short hairpin (sh)RNA to PC3 cells for advanced PCa therapy.

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p = 0.003) and 54% (p = 0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p = 0.001), MYOG (94%, p = 0.000), and MYF5 (36%, p = 0.000), cell cycle regulator p21 (62%, p = 0.000), MSTN receptor ACVR2B (23%, p = 0.061), MSTN antagonist follistatin (81%, p = 0.000), and downstream signaling mediators SMAD2 (20%, p = 0.060) and SMAD3 (49%, p = 0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p = 0.354) and 79% compared to untransduced myoblasts (p = 0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p = 0.000)- and sh2 (1.71-fold; p = 0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.

Lsd1 Restricts the Number of Germline Stem Cells by Regulating Multiple Targets in Escort Cells

Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC) phenotype. Here we present evidence that loss of Lsd1 also results in gradual changes in escort cell morphology and their eventual death. To better characterize the function of Lsd1 in different cell populations within the ovary, we performed Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq). This analysis shows that Lsd1 associates with a surprisingly limited number of sites in escort cells and fewer, and often, different sites in cap cells. These findings indicate that Lsd1 exhibits highly selective binding that depends greatly on specific cellular contexts. Lsd1 does not directly target the dpp locus in escort cells. Instead, Lsd1 regulates engrailed expression and disruption of engrailed and its putative downstream target hedgehog suppress the Lsd1 mutant phenotype. Interestingly, over-expression of engrailed, but not hedgehog, results in an expansion of GSC cells, marked by the expansion of BMP signaling. Knockdown of other potential direct Lsd1 target genes, not obviously linked to BMP signaling, also partially suppresses the Lsd1 mutant phenotype. These results suggest that Lsd1 restricts the number of GSC-like cells by regulating a diverse group of genes and provide further evidence that escort cell function must be carefully controlled during development and adulthood to ensure proper germline differentiation.

Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.

Target Gene Knockdown by 2′,4′-BNA/LNA Antisense Oligonucleotides in Zebrafish

Gene knockdowns using oligonucleotide-based approaches are useful for studying gene function in both in vitro cell culture systems and in vivo animal models. We evaluated the efficacy of 2',4'-bridged nucleic acids (BNA)-modified antisense oligonucleotides (AONs) for gene knockdown in zebrafish. We used the tcf7l1a gene as a model for testing the knockdown efficacy of 2',4'-BNA AONs and examined how the target sites/affinity and RNase H induction activity of 2',4'-BNA AONs affect knockdown efficacy. We found that tcf7l1a gene function was knocked down by 2',4'-BNA AONs that target the start codon and induce RNase H activity. Although nonspecific p53-mediated developmental defects were observed at higher doses, the effective dose of the 2',4'-BNA AONs for tcf7l1a is much lower than that of morpholino oligonucleotides. Our data thus show a potential application for 2',4'-BNA AONs in the downregulation of specific genes in zebrafish.

Derivation and manipulation of trophoblast stem cells from mouse blastocysts.

The trophoblast is the first lineage to undergo differentiation during mammalian development. In the preimplantation blastocyst embryo, two cell types are present including the inner cell mass (ICM) and the trophectoderm (TE). ICM cells exhibit pluripotent potential, or the capacity to give rise to all cells represented in the adult organism, while TE cells are multipotent and are therefore only capable of differentiating into trophoblast lineages represented in the placenta. The TE is essential for implantation of the embryo into the uterine tissue, formation of trophoblast lineages represented in the placenta, and exchange of nutrients and waste between the embryo and the mother. Trophoblast stem (TS) cells, which can be derived from the TE of preimplantation embryos in the presence of external signals such as FGF4, can self-renew indefinitely, and because they are capable of differentiating into epithelial lineages of the trophoblast, TS cells are a useful in vitro model to study the biology of the trophoblast including epigenetic regulation of gene expression. In this chapter we describe protocols for derivation of TS cells from mouse blastocysts, culture conditions that promote self-renewal and differentiation, and methods to transduce TS cells with lentiviral particles encoding shRNAs. These protocols are sufficient for efficient derivation of TS cells and robust RNAi knockdown of target genes in TS cells.

Advances in Genome Engineering Approaches

Targeted genome editing is essential for functional characterization of a gene of interest. Targeted gene inactivation via homologous recombination made it feasible to create gene knockout animal models to ascertain the physiological role of the target genes; however, lower efficiency of site specific insertion of the genetically modified construct through homologous recombination has limited a wider applicability of this approach. Development of targeted gene knockdown through RNA interferce (RNAi) offered a cost effective, high-throughput alternative to homologous recombination, however, RNAi-mediated gene knockdown is incomplete, produces experiment to experiment variation, and could provide only a temporary inhibition of the gene function. Development of genome engineering methodologies utilizing nucleases linked to the guide sequences targeting a gene of interest, such as Zinc Finger Nucleases (ZFN), Transcription Activator like Effector Nucleases (TALEN) and Clustered Palindromic Repeats (CRISPR), are quite encouraging. A brief overview of recent advances in genome engineering approaches is provided with their respective advantages and limitations.

Triple Combination of siRNAs Targeting TGFβ1, TGFβR2, and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts

Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA). Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry. Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.

Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E–dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Enhancing cell growth and antibody production in CHO cells by siRNA knockdown of novel target genes

Background Seven out of the ten top-selling biopharmaceuticals in 2011 are produced in Chinese Hamster Ovary (CHO) cells [1]. This tremendous commercial interest makes the development and application of strategies for cell line optimization, like gene overexpression or knockdown to enhance cell specific productivity and cellular growth, highly interesting. In this work, we investigated the knockdown effect of novel target genes by siRNA as a powerful tool for CHO cell line engineering.

Abstract B222: Dicer substrate siRNAs to MYC, B-catenin, and other target genes effectively induce in vivo target gene knockdown and tumor inhibition.

Abstract Although there are now multiple successful examples of targeted therapeutics in cancer, many key protein targets have remained largely ‘undruggable’, including transcription factors such as B-catenin (CTNNB1) and MYC. B-catenin mediates Wnt signaling, which is overactive in many cancers including hepatocarcinoma (HCC). RNAi offers a way to reach such undruggable targets by inhibiting their expression at the mRNA level. Dicer substrate siRNAs (“DsiRNAs”) can be particularly effective for gene silencing. DsiRNAs are longer than conventional siRNAs, and therefore are substrates for processing by Dicer, after which the product RNA duplexes are incorporated into RISC, leading to target mRNA knockdown. We have used DsiRNAs to target B-catenin, MYC, and other key genes in HCC and other cancers. Through large-scale DsiRNA screening, we have identified a series of high potency DsiRNAs with picomolar to sub-picomolar IC50 values for mRNA knockdown. Lead DsiRNAs fully tolerated extensive 2’-OMe modification, retaining high potency, and lacked detectable immunostimulatory activity. To test the effect of target gene knockdown on tumor growth in vivo, we used a lipid nanoparticle (LNP) delivery system. LNP/DsiRNA particles effectively delivered to tumors in mouse tumor models, leading to rapid knockdown of target gene mRNA and protein. Knockdown of B-catenin, MYC, and other target genes strongly inhibited tumor growth. B-catenin knockdown also strongly reduced expression of the B-catenin-regulated genes Axin2 and MYC, a potential mechanism for tumor inhibition. In summary, we have developed high potency DsiRNAs to cancer target genes, and effectively delivered these DsiRNAs to tumors in vivo, resulting in inhibition of target gene expression and inhibition of tumor growth. DsiRNA therapeutics show promise as novel agents for reaching traditionally undruggable target genes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B222. Citation Format: Hank Dudek, Kathleen Wortham, Rokhand Arvan, Anee Shah, Bo Ying, Wendy Cyr, Hailin Yang, Wei Zhou, Utsav Saxena, Yi Zhou, Rohan Diwanji, Ben Holmes, Ruchir Farkiwala, Aalok Shah, Bob Brown. Dicer substrate siRNAs to MYC, B-catenin, and other target genes effectively induce in vivo target gene knockdown and tumor inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B222.

Abstract B5: A BMP7 variant inhibits angiogenesis in vitro and in vivo in part by downregulating VEGFR2 and FGFR1 expression in endothelial cells.

Abstract Glioblastoma multiforme (GBM), the most aggressive glioma, requires active angiogenesis for growth and survival. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Previously, we demonstrated the use of a BMP7 variant (BMP7v) to differentiate glioblastoma stem-like cells (GSLCs) and significantly reduce their tumorigenic potential (Tate and Pallini et al. 2012). Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, and its cognate in vivo model, we investigated the role of BMP7v in VEGF, basic FGF (bFGF), tumor-driven angiogenesis. Furthermore, we explored the effect of BMP7v on angiogenesis in GSLC Matrigel plugs to further establish the relevance of the BMP7 effect in a pathological setting. BMP7v treatment resulted in disruption of neo-endothelial cord formation and regression of existing bFGF established cords in vitro. In addition, pre-treatment of endothelial cells with BMP7v led to a significant reduction in their cord forming ability, indicating a direct effect of BMP7v on endothelial cell function. While BMP7v activated the canonical Smad signaling pathway in endothelial cells, targeted gene knockdown using shRNA directed against Smad 4 suggested this pathway is not required to mediate the anti-angiogenic effect of BMP7v. BMP7v decreased endothelial cell migration and down regulated expression of the receptor tyrosine kinases involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1, respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of functional angiogenesis, BMP7v significantly decreased hemoglobin content. In addition, BMP7v significantly decreased angiogenesis in GSLC Matrigel plugs indicating efficacy in a more relevant cancer setting. These data support BMP7v as a potent anti-angiogenic molecule, and the reduction in GSLC tumorigenicity may be due in part to reduced angiogenesis upon BMP7v treatment. Future experiments will attempt to further define the mechanism underlying BMP7v's anti-angiogenic effect. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B5. Citation Format: Courtney Tate, Jacquelyn McEntire, Roberto Pallini, Lisa Wyss, Wayne Blosser, Eliza Vakana, Giorgio D'Alessandris, Liliana Morgante, Stefano Giannetti, Luigi Larocca, Giorgio Stassi, Antonina Benfante, Maria Colorito, Ruggero De Maria, Scott Rowlinson, Louis Stancato. A BMP7 variant inhibits angiogenesis in vitro and in vivo in part by downregulating VEGFR2 and FGFR1 expression in endothelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B5.

Highlighting the Role of Polymer Length, Carbohydrate Size, and Nucleic Acid Type in Potency of Glycopolycation Agents for pDNA and siRNA Delivery

While nucleic acids such as small interfering RNA (siRNA) and plasmid DNA (pDNA) are promising research tools and therapeutic modalities, their potential in medical applications is limited by a fundamental mechanistic understanding and inadequate efficiency. Herein, two series of carbohydrate-based polycations were synthesized and examined that varied in the degree of polymerization (n), one containing trehalose [Tr4(n) series: Tr4(23), Tr4(55), Tr4(77)] and the other containing β-cyclodextrin [CD4(n) series: CD4(10), CD4(26), CD4(39), CD4(143), CD4(239)]. In addition, two monosaccharide models were examined for comparison that contain tartaramidoamine (T4) and galactaramidoamine (G4 or Glycofect) repeats. Delivery profiles for pDNA were compared with those obtained for siRNA delivery and reveal that efficacy differs significantly as a function of carbohydrate type, nucleic acid type and dose, polymer length, and presence of excess polymer in the formulation. The Tr4 polymers yielded higher efficacy for pDNA delivery, yet the CD4 polymers achieved higher siRNA delivery and gene down-regulation. The T4 and Glycofect derivatives, while efficient for pDNA delivery, were completely ineffective for siRNA delivery. A strong polymer length and dose dependence on target gene knockdown was observed for all polymers tested. Also, free polymer in solution (uncomplexed) was demonstrated to be a key factor in promoting siRNA uptake and gene down-regulation.

Chemical modifications on siRNAs avoid Toll-like-receptor-mediated activation of the hepatic immune system in vivo and in vitro

The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off-target effects, especially in the liver. Therefore, we assessed the potential of differently modified siRNAs to induce the hepatic innate immune system in vitro and in vivo. Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6-48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR. Unmodified GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-β and IFN-sensitive gene 15 in vivo, whereas a formulation of 2'-O-methylated-LUC siRNA had no such effects. Formulation of unmodified APOB1-specific siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48h after application. The results were paralleled in vitro, where transfection of liver cells with unmodified siRNAs, but not with chemically modified siRNAs, led to cell-type-specific induction of immune genes. These immune responses were not observed in MYD88-deficient mice or in chloroquine-treated cells in vitro. Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2'-O-methyl modifications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics.