SLN124, a GalNAc conjugated 19-mer siRNA targeting tmprss6, reduces plasma iron and increases hepcidin levels of healthy volunteers.

SLN124, an N-acetylgalactosamine conjugated 19-mer short interfering RNA, is being developed to treat iron-loading anemias (including beta-thalassemia and myelodysplastic syndromes) and myeloproliferative neoplasms (polycythemia vera). Through hepatic targeting and silencing of the TMPRSS6 gene, SLN124 increases endogenous hepcidin synthesis. This is the first clinical report of an siRNA targeting a component of iron homeostasis. This first-in-human, phase 1 study assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending doses of SLN124 (1.0, 3.0, and 4.5 mg/kg) in healthy volunteers. Twenty-four participants were randomized in three sequential cohorts of eight subjects, each to receive a single dose of either SLN124 or placebo (6:2 randomization), administered subcutaneously. There were no serious or severe adverse events, or discontinuations due to adverse events, and most treatment-emergent adverse events were mild, including transient mild injection site reactions, resolving without intervention. SLN124 was rapidly absorbed, with a median tmax of 4-5 h across all treatment groups, and largely eliminated from plasma by 48 h. Plasma concentrations increased in a greater than dose proportional fashion between treatment groups. In all SLN124 groups, a dose-related effect was observed across iron metabolism markers, and across erythroid markers, SLN124 resulted in increased plasma hepcidin levels, peaking around Day 29, and consequent dose-related sustained reductions in plasma iron and transferrin saturation with decreased reticulocyte production, MCHC, and MCV. Results suggest duration of action lasting up to 56 days after a single SLN124 dose, on hepcidin and hematological parameters of iron metabolism (serum iron and TSAT).

Pre-clinical assessment of SLN360, a novel siRNA targeting LPA, developed to address elevated lipoprotein (a) in cardiovascular disease.

BACKGROUND AND AIMS: The LPA gene encodes apolipoprotein (a), a key component of Lp(a), a potent risk factor for cardiovascular disease with no specific pharmacotherapy. Here we describe the pharmacological data for SLN360, a GalNAc-conjugated siRNA targeting LPA, designed to address this unmet medical need. METHODS: SLN360 was tested in vitro for LPA knockdown in primary hepatocytes. Healthy cynomolgus monkeys received single or multiple subcutaneous doses of the SLN360 sequence ranging from 0.1 to 9.0 mg/kg to determine the pharmacokinetic and pharmacodynamic effects. Liver mRNA and serum biomarker analyses were performed. RESULTS: In vitro, the SLN360 sequence potently reduces LPA mRNA in primary cynomolgus and human hepatocytes, while no effect was observed on the expression of APOB or PLG. In vivo, SLN360 exposure peaks 2 h after subcutaneous injection with near full elimination by 24 h. Specific LPA mRNA reduction (up to 91% 2 weeks after dosing) was observed with only the 3 mg/kg group showing appreciable return to baseline (40%). No consistent dose- or time-dependent effect on the expression of APOB, PLG or a panel of sensitive markers of liver lipid accumulation was observed. Potent (up to 95%) and long lasting (≥9 weeks) serum Lp(a) reduction was observed, peaking in all active groups at day 21. The minimally effective dose was determined to be 0.3 mg/kg with an ED50 of 0.6 mg/kg. CONCLUSIONS: SLN360 induces a sustained reduction in serum Lp(a) levels in cynomolgus monkeys following subcutaneous dosing. SLN360 has potential to address the unmet need of Lp(a) reduction in cardiovascular diseases.

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.

Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues.

Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.

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.