Delivery Systems for the Direct Application of siRNAs to Induce RNA Interference (RNAi) In Vivo

RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described

Polymeric Nanoparticles Based on Tyrosine-Modified, Low Molecular Weight Polyethylenimines for siRNA Delivery

A major hurdle for exploring RNA interference (RNAi) in a therapeutic setting is still the issue of in vivo delivery of small RNA molecules (siRNAs). The chemical modification of polyethylenimines (PEIs) offers a particularly attractive avenue towards the development of more efficient non-viral delivery systems. Here, we explore tyrosine-modified polyethylenimines with low or very low molecular weight (P2Y, P5Y, P10Y) for siRNA delivery. In comparison to their respective parent PEI, they reveal considerably increased knockdown efficacies and very low cytotoxicity upon tyrosine modification, as determined in different reporter and wildtype cell lines. The delivery of siRNAs targeting the anti-apoptotic oncogene survivin or the serine/threonine-protein kinase PLK1 (polo-like kinase 1; PLK-1) oncogene reveals strong inhibitory effects in vitro. In a therapeutic in vivo setting, profound anti-tumor effects in a prostate carcinoma xenograft mouse model are observed upon systemic application of complexes for survivin or PLK1 knockdown, in the absence of in vivo toxicity. We thus demonstrate the tyrosine-modification of (very) low molecular weight PEIs for generating effcient nanocarriers for siRNA delivery in vitro and in vivo, present data on their physicochemical and biological properties, and show their efficacy as siRNA therapeutic in vivo, in the absence of adverse effects

Inhibition of HER Receptors Reveals Distinct Mechanisms of Compensatory Upregulation of Other HER Family Members: Basis for Acquired Resistance and for Combination Therapy

Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors

Tyrosine-Modification of Polypropylenimine (PPI) and Polyethylenimine (PEI) Strongly Improves Efficacy of siRNA-Mediated Gene Knockdown

The delivery of small interfering RNAs (siRNA) is an efficient method for gene silencing through the induction of RNA interference (RNAi). It critically relies, however, on efficient vehicles for siRNA formulation, for transfection in vitro as well as for their potential use in vivo. While polyethylenimines (PEIs) are among the most studied cationic polymers for nucleic acid delivery including small RNA molecules, polypropylenimines (PPIs) have been explored to a lesser extent. Previous studies have shown the benefit of the modification of small PEIs by tyrosine grafting which are featured in this paper. Additionally, we have now extended this approach towards PPIs, presenting tyrosine-modified PPIs (named PPI-Y) for the first time. In this study, we describe the marked improvement of PPI upon its tyrosine modification, leading to enhanced siRNA complexation, complex stability, siRNA delivery, knockdown efficacy and biocompatibility. Results of PPI-Y/siRNA complexes are also compared with data based on tyrosine-modified linear or branched PEIs (LPxY or PxY). Taken together, this establishes tyrosine-modified PPIs or PEIs as particularly promising polymeric systems for siRNA formulation and delivery

Anti-tumor effects of fibroblast growth factor-binding protein (FGF-BP) knockdown in colon carcinoma

Abstract 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.</p

Anticancer Therapy with HDAC Inhibitors: Mechanism-Based Combination Strategies and Future Perspectives

The increasing knowledge of molecular drivers of tumorigenesis has fueled targeted cancer therapies based on specific inhibitors. Beyond “classic” oncogene inhibitors, epigenetic therapy is an emerging field. Epigenetic alterations can occur at any time during cancer progression, altering the structure of the chromatin, the accessibility for transcription factors and thus the transcription of genes. They rely on post-translational histone modifications, particularly the acetylation of histone lysine residues, and are determined by the inverse action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Importantly, HDACs are often aberrantly overexpressed, predominantly leading to the transcriptional repression of tumor suppressor genes. Thus, histone deacetylase inhibitors (HDACis) are powerful drugs, with some already approved for certain hematological cancers. Albeit HDACis show activity in solid tumors as well, further refinement and the development of novel drugs are needed. This review describes the capability of HDACis to influence various pathways and, based on this knowledge, gives a comprehensive overview of various preclinical and clinical studies on solid tumors. A particular focus is placed on strategies for achieving higher efficacy by combination therapies, including phosphoinositide 3-kinase (PI3K)-EGFR inhibitors and hormone- or immunotherapy. This also includes new bifunctional inhibitors as well as novel approaches for HDAC degradation via PROteolysis-TArgeting Chimeras (PROTACs)

Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition

Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise -cyclodextrinmodified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53 /PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities

Analysis of cellular and molecular antitumor effects upon inhibition of SATB1 in glioblastoma cells

Background: The Special AT-rich Sequence Binding Protein 1 (SATB1) regulates the expression of many genes by acting as a global chromatin organizer. While in many tumor entities SATB1 overexpression has been observed and connected to pro-tumorigenic processes, somewhat contradictory evidence exists in brain tumors with regard to SATB1 overexpression in glioblastoma and its association with poorer prognosis and tumor progression. On the functional side, initial data indicate that SATB1 may be involved in several tumor cell-relevant processes. Methods: For the detailed analysis of the functional relevance and possible therapeutic potential of SATB1 inhibition, we employ transient siRNA-mediated knockdown and comprehensively analyze the cellular and molecular role of SATB1 in glioblastoma. Results: In various cell lines with different SATB1 expression levels, a SATB1 gene dose-dependent inhibition of anchorage-dependent and –independent proliferation is observed. This is due to cell cycle-inhibitory and pro-apoptotic effects of SATB1 knockdown. Molecular analyses reveal SATB1 knockdown effects on multiple important (proto-) oncogenes, including Myc, Bcl-2, Pim-1, EGFR, β-catenin and Survivin. Molecules involved in cell cycle, EMT and cell adhesion are affected as well. The putative therapeutic relevance of SATB1 inhibition is further supported in an in vivo tumor xenograft mouse model, where the treatment with polymeric nanoparticles containing SATB1-specific siRNAs exerts antitumor effects. Conclusion: Our results demonstrate that SATB1 may represent a promising target molecule in glioblastoma therapy whose inhibition or knockdown affects multiple crucial pathways