Search

MIR143 is pathologically downregulated and may function as a tumor suppressor in prostate cancer. Likewise, the urokinase plasminogen activator receptor (UPAR) is overexpressed in prostate carcinoma, representing a negative prognostic marker and putative therapeutic target gene. In this paper, we establish UPAR as a new direct target of MIR143. Luciferase reporter gene constructs identify one of the two in silico-predicted binding sites as functionally relevant for direct MIR143 binding to the 3′ UTR, and, concomitantly, transfection of MIR143 reduces UPAR protein levels in prostate carcinoma cells in vitro. Inhibitory effects on cell proliferation and colony formation, spheroid growth and integrity, and cell viability are extensively analyzed, and they are compared to direct small interfering RNA (siRNA)-mediated uPAR knockdown or combined microRNA (miRNA)-siRNA treatment. Switching to a therapeutically more relevant in vivo model, we demonstrate tumor-inhibitory effects of MIR143 replacement therapy by systemic treatment of mice bearing subcutaneous PC-3 tumor xenografts with MIR143 formulated in polymeric nanoparticles. This efficient, nanoparticle-mediated delivery of intact MIR143 mediates the marked downregulation of uPAR protein, but not mRNA levels, thus indicating translational inhibition rather than mRNA degradation. In summary, we identify UPAR as a direct target gene of MIR143, and we establish the therapeutic anti-tumor potential of nanoparticle-based MIR143 replacement in prostate cancer. Keywords: MIR143, urokinase plasminogen activator receptor, prostate cancer, miRNA replacement, PEI nanoparticles, xenograft

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson’s disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4–12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson’s disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention., Graphical Abstract

// Rishein Gupta 1,* , Tanvi Arkatkar 1,* , Jonathon Keck 1 , Gopala Krishna Lanka Koundinya 1 , Kevin Castillo 1 , Sabrina Hobel 2 , James P. Chambers 1 , Jieh-Juen Yu 1 , M. Neal Guentzel 1 , Achim Aigner 2 , Lane K. Christenson 3 and Bernard P. Arulanandam 1 1 South Texas Center for Emerging Infectious Diseases and Center of Excellence in Infection Genomics, University of Texas at San Antonio, San Antonio, TX, USA 2 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Hartelstrase, Leipzig, Germany 3 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA * These authors have contributed equally to this work Correspondence to: Bernard P. Arulanandam, email: // Keywords : Chlamydia muridarum , host immunity, microRNA-155, microRNA-182, CD4+ T-cells, Immunology and Microbiology Section, Immune response, Immunity Received : July 22, 2016 Accepted : August 02, 2016 Published : August 20, 2016 Abstract Anti-chlamydial immunity involves efficient presentation of antigens (Ag) to effector cells resulting in Ag-specific immune responses. There is limited information on inherent underlying mechanisms regulating these events. Previous studies from our laboratory have established that select microRNAs (miRs) function as molecular regulators of immunity in Chlamydia muridarum (Cm) genital infection. In this report, we investigated immune cell type-specific miRs, i.e. miR-155 and -182, and the role in Ag-specific immunity. We observed significant up-regulation of miR-155 in C57BL/6 bone marrow derived dendritic cells (BMDC), and miR-182 in splenic Ag-specific CD4 + T-cells. Using mimics and inhibitors, we determined that miR-155 contributed to BMDC activation following Cm infection. Co-cultures of miR-155 over-expressed in BMDC and miR-182 over-expressed in Ag-specific CD4 + T-cells, or miR-155 -/- BMDC with miR-182 inhibitor treated Ag-specific CD4 + T-cells, resulted in IFN-γ production comparable to Ag-specific CD4 + T-cells isolated from Cm infected mice. Additionally, miR-182 was significantly up-regulated in intranasally vaccinated mice protected against Cm infection. In vivo depletion of miR-182 resulted in reduction in Ag-specific IFN-γ and genital pathology in Cm infected mice. To the best of our knowledge, this is the first study to report an interaction of miR-155 (in Cm infected DC) and miR-182 (in CD4 + T-cell) resulting in Ag specific immune responses against genital Cm.

Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.

RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.

Intraperitoneal (IP) administration of nano-sized delivery vehicles containing small interfering RNA (siRNA) has recently gained attention as an alternative route for the efficient treatment of peritoneal carcinomatosis. The colloidal stability of nanomatter following IP administration has, however, not been thoroughly investigated yet. Here, enabled by advanced microscopy methods such as single particle tracking and fluorescence correlation spectroscopy, we follow the aggregation and cargo release of nano-scaled systems directly in peritoneal fluids from healthy mice and ascites fluid from a patient diagnosed with peritoneal carcinomatosis. The colloidal stability in the peritoneal fluids was systematically studied as a function of the charge (positive or negative) and poly(ethylene glycol) (PEG) degree of liposomes and polystyrene nanoparticles, and compared to human serum. Our data demonstrate strong aggregation of cationic and anionic nanoparticles in the peritoneal fluids, while only slight aggregation was observed for the PEGylated ones. PEGylated liposomes, however, lead to a fast and premature release of siRNA cargo in the peritoneal fluids. Based on our observations, we reflect on how to tailor improved delivery systems for IP therapy.

Background Fibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM). One mechanism of their bioactivation relies on the FGF-binding protein (FGF-BP) which, upon reversible binding to FGF-1 or -2, leads to their release from the ECM. FGF-BP increases tumorigenicity and is highly expressed in tumors like colon carcinoma. In this paper, we analyse cellular and molecular consequences of RNAi-mediated FGF-BP knockdown in colon carcinoma, and explore the therapeutic effects of the nanoparticle-mediated delivery of small interfering RNAs (siRNAs) for FGF-BP targeting. Results Employing stable RNAi cells, we establish a dose-dependence of cell proliferation on FGF-BP expression levels. Decreased proliferation is mirrored by alterations in cell cycle distribution and upregulation of p21, which is relevant for mediating FGF-BP effects. While inhibition of proliferation is mainly associated with reduced Akt and increased GSK3β activation, antibody array-based analyses also reveal other alterations in MAPK signalling. Additionally, we demonstrate induction of apoptosis, mediated through caspase-3/7 activation, and alterations in redox status upon FGF-BP knockdown. These effects are based on the upregulation of Bad, Bax and HIF-1α, and the downregulation of catalase. In a therapeutic FGF-BP knockdown approach based on RNAi, we employ polymer-based nanoparticles for the in vivo delivery of siRNAs into established wildtype colon carcinoma xenografts. We show that the systemic treatment of mice leads to the inhibition of tumor growth based on FGF-BP knockdown. Conclusions FGF-BP is integrated in a complex network of cytoprotective effects, and represents a promising therapeutic target for RNAi-based knockdown approaches.

The efficient delivery of nucleic acids into cells is critical for successful gene therapy or gene knockdown. Polyethylenimines (PEIs) are positively charged polymers which complex and deliver DNA for gene transfection or small interfering RNAs (siRNAs) for the induction of RNA interference (RNAi), and mediate their endosomal release. Likewise, various liposomes act as transfection reagents, with some lipids showing increased endocytosis and influencing endosomal escape. This study combines the favourable properties of PEI and lipid systems for DNA and siRNA delivery. Various lipids and lipid combinations, which cover a broad range of physicochemical properties and form optimal liposomes, are assessed. By addition of the liposomes to pre-formed polyplexes, based on the low molecular weight PEI F25-LMW, we establish liposome-PEI complexes (lipopolyplexes) and characterise them in comparison to their 'parent' polyplexes and liposomes regarding size, shape and zeta-potential. Furthermore, while the lipidation of polyplexes generally decreases their toxicity, our studies on DNA transfection and siRNA-mediated knockdown also establish certain lipopolyplexes based on rigid, negatively charged lipids as particularly efficient vehicles for nucleic acid delivery, further improving DNA transfection. The analysis of their mechanism and kinetics of cellular uptake confirms that the biological properties of lipopolyplexes are mainly determined by the liposome shell. We conclude that certain lipopolyplexes show improved biological properties over PEI complexes, thus representing potentially attractive non-viral vectors for gene therapy and RNAi.

RNA interference (RNAi) is a powerful strategy to inhibit gene expression through specific mRNA degradation mediated by small interfering RNAs (siRNAs). In vivo, however, the application of siRNAs is severely limited by their instability and poor delivery into target cells and target tissues. Glioblastomas are the most frequent and malignant brain tumors with, so far, limited treatment options. To develop novel and more efficacious therapies, advanced targeting strategies against glioblastoma multiforme (GBM)-relevant target genes must be established in vivo. Here we use RNAi-based targeting of the secreted growth factor pleiotrophin (PTN), employing a polyethylenimine (PEI)/siRNA complex strategy. We show that the complexation of chemically unmodified siRNAs with PEI leads to the formation of complexes that condense and completely cover siRNAs as determined by atomic force microscopy (AFM). On the efficient cellular delivery of these PEI/siRNA complexes, the PTN downregulation in U87 glioblastoma cells in vitro results in decreased proliferation and soft agar colony formation. More importantly, in vivo treatment of nude mice through systemic application (subcutaneous or intraperitoneal) of PEI-complexed PTN siRNAs leads to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth without a measurable induction of siRNA-mediated immunostimulation. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerts antitumoral effects. In conclusion, we present the PEI complexation of siRNAs as a universally applicable platform for RNAi in vitro and in vivo and establish, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM.

The therapeutic application of RNA interference (RNAi) for the knockdown of tumor-relevant genes critically relies on the efficient delivery of small interfering RNAs (siRNAs) or shRNA-encoding DNA plasmids in vivo. Previously, we have established polyethylenimine (PEI)/siRNA complexes as a highly efficient and ease-to-use system for gene targeting and a universally applicable platform for therapeutic RNAi. Here, we analyse the anti-tumor effects of the therapeutic knockdown of various members of the HER family, i.e. HER1 (c-erbB1, EGFR), HER2 (c-erbB2/neu) and HER3 (c-erbB3) in s.c. ovarian carcinoma xenografts in mice. While little effects are seen upon HER1 knockdown, the treatment of the mice with PEI/complexed siRNAs targeting HER2 or HER3 lead to a significant reduction in tumor growth. Notably, additive anti-tumor effects are observed upon double knockdown of HER2 and HER3. The biocompatibility of this PEI-based siRNA delivery platform for in vivo RNAi can be further increased by modifications of the polyethylenimines. To this end, we systematically analyse physicochemical and biological properties of DNA and siRNA complexes prepared from a set of maltose-, maltotriose- or maltoheptaose-modified hyperbranched PEIs (termed (oligo-)maltose-modified PEIs; OM-PEIs). While (oligo-)maltose grafting generally leads to reduced in vitro DNA transfection efficacies, this effect is less profound in OM-PEI/siRNA complexes for the induction of RNAi. This is particularly true for NH2-monosubstituted OM-PEIs. Independent of the maltose architecture, DNA and siRNA complexes based on maltose-grafted PEI show considerably lower toxicity as compared to PEI complexes. More importantly, pharmacokinetics in mice are markedly altered in OM-PEI/siRNA complexes. While their halflives in the blood circulation upon i.v. injection are generally increased as compared to PEI/siRNA complexes, the tissue distribution of radioactively labelled (OM-)PEI complexed siRNA molecules critically relies on the degree and pattern of (oligo-)maltose grafting. Marked differences in siRNA tissue distribution profiles are observed between different OM-PEIs. Enhanced tissue siRNA levels upon treatment of mice with OM-PEI/siRNA complexes as compared to PEI-based complexes are observed. Taken together, we (i) demonstrate the therapeutic anti-tumor effects of PEI/siRNA-mediated (double) knockdown of members of the HER family and (ii) establish OM-PEI complexes as novel promising tools for the in vivo application of siRNAs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 594.