Cationic LNP-formulated mRNA expressing Tie2-agonist in the lung endothelium prevents pulmonary vascular leakage.

Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model.

First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors.

PURPOSE: Atu027 is a novel liposomal RNA interference therapeutic that includes a short-interfering RNA (siRNA), which silences expression of protein kinase N3 in the vascular endothelium. Atu027 has previously been shown to inhibit local tumor invasion as well as lymph node and pulmonary metastasis in mouse cancer models. This first-in-human study aimed to assess the safety, tolerability, and pharmacokinetics of Atu027 while evaluating therapeutic effects on both primary tumors and metastatic lesions. PATIENTS AND METHODS: Thirty-four patients with advanced solid tumors received 10 escalating doses of Atu027 without premedication, as one single followed by eight intravenous infusions twice per week during a 28-day cycle. Response was monitored by computed tomography/magnetic resonance imaging at baseline, at the end of treatment (EoT), and at final follow-up (EoS), and was assessed according to RECIST. RESULTS: Atu027 was well tolerated up to dose levels of 0.336 mg/kg; most adverse events (AEs) were low-grade toxicities (grade 1 or 2). No maximum tolerated dose was reached. Plasma levels of siRNA strands and lipids were dose proportional, peaking during 4-hour infusion. Disease stabilization was achieved in 41% of patients at EoT (n = 14 of 34 treated patients); eight patients had stable disease at EoS, and some experienced complete or partial regression of metastases. sFLT1 (soluble variant of vascular endothelial growth factor receptor-1) decreased from pretreatment levels in most patients after dose levels 04 to 10. CONCLUSION: Atu027 was safe in patients with advanced solid tumors, with 41% of patients having stable disease for at least 8 weeks. In view of these results, further clinical trials have been initiated, and sFLT1 will be investigated as a potential biomarker.

Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models.

PURPOSE: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models. EXPERIMENTAL DESIGN: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results. RESULTS: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation. CONCLUSION: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy.

Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression.

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density.

REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase.

Cancer cells frequently evade apoptosis during tumorigenesis by acquiring mutations in apoptotic regulators. Chronic activation of the PI 3-kinase-Akt pathway through loss of the tumor suppressor PTEN is one mechanism by which these cells can gain increased protection against apoptosis. We report here that REDD1 (RTP801) can act as a transcriptional downstream target of PI 3-kinase signaling in human prostate cancer cells (PC-3). REDD1 expression is markedly reduced in PC-3 cells treated with LY294002 (LY) or Rapamycin and strongly induced under hypoxic conditions in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. Loss of function studies employing antisense molecules or RNA interference indicate that REDD1 is essential for invasive growth of prostate cancer cells in vitro and in vivo. Reduced REDD1 levels can sensitize cells towards apoptosis, whereas elevated levels of REDD1 induced by hypoxia or overexpression desensitize cells to apoptotic stimuli. Taken together our data designate REDD1 as a novel target for therapeutic intervention in prostate cancer.

PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase.

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

Knockdown of MTP18, a novel phosphatidylinositol 3-kinase-dependent protein, affects mitochondrial morphology and induces apoptosis.

We identified a novel human cDNA encoding a mitochondrial protein, MTP18 (mitochondrial protein, 18 kDa) as a transcriptional downstream target of phosphatidylinositol (PI) 3-kinase signaling. We demonstrate that MTP18 mRNA as well as protein expression is dependent on PI 3-kinase activity. Confocal microscopy and biochemical fractionation revealed a mitochondrial localization of MTP18. Loss-of-function analysis employing antisense molecules revealed that MTP18 is essential for cell viability in PC-3 and HaCaT cells. We show that knockdown of MTP18 protein level results in a cytochrome c release from mitochondria and consequently leads to apoptosis. In addition, HaCaT cells with reduced levels of MTP18 become more sensitive to apoptotic stimuli. This effect is accompanied by dramatic subcellular alterations. Reduction of MTP18 impairs mitochondrial morphology resulting in the formation of a highly interconnected mitochondrial reticulum in COS-7 cells. Conversely, overexpression of MTP18 induces a punctuate morphology of mitochondria suggesting also a functional role of MTP18 in maintaining the mitochondrial integrity. Hence, our data indicate an unexpected connection of PI 3-kinase signaling, apoptosis and the morphology of mammalian mitochondria.

Inducible shRNA expression for application in a prostate cancer mouse model.

RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.

Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.

Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA.

RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110beta, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.

GeneBlocs are powerful tools to study and delineate signal transduction processes that regulate cell growth and transformation.

The study of signal transduction processes using antisense oligonucleotides is often complicated by low intracellular stability of the antisense reagents or by nonspecific effects that cause toxicity. Here, we introduce a new class of antisense molecules, so-called GeneBlocs, which are characterized by improved stability, high target RNA specificity, and low toxicity. GeneBlocs allow for efficient downregulation of mRNA expression at nanomolar concentrations, and they do not interfere with cell proliferation. We demonstrate these beneficial properties using a positive readout system. GeneBloc-mediated inhibition of tumor suppressor PTEN (phosphatase and tension homologue detected on chromosome 10) expression leads to hyperactivation of the phosphatidylinositol (PI) 3-kinase pathway, thereby mimicking the loss of PTEN function and its early consequences observed in mammalian cancer cells. Specifically, cells treated with PTEN GeneBlocs show functional activation of Akt, a downstream effector of PI 3-kinase signaling, and exhibit enhanced proliferation when seeded on a basement membrane matrix. In addition, GeneBlocs targeting the catalytic subunit of PI 3-kinase, p110, specifically inhibit signal transduction of endogenous or recombinant PI 3-kinase. This demonstrates that GeneBlocs are powerful tools to analyze and to modulate signal transduction processes and, therefore, represent alternative reagents for the validation of gene function.

Unravelling novel intracellular pathways in cell-based assays.

The pharmaceutical industry is currently facing several challenges to identify and develop novel drug targets. Traditional drug discovery focussed on a small number of well-characterized gene products. Recently, this picture has changed with the completion of the draft sequence of the human genome, which has led to the identification of thousands of novel genes with unknown or poorly understood function. To cope with this overwhelming number of potential drug target candidates, new strategies for the elucidation of gene function, as well as their involvement in intracellular pathways, are required.