We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

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Recent studies have consistently found pregnancy-associated plasma protein A2 (PAPP-A2) to be upregulated in preeclamptic placentae at term. We tested whether first-trimester circulating PAPP-A2 levels differed between complicated and uncomplicated pregnancies. We measured maternal PAPP-A2 levels at 10 to 14 weeks of gestational age in 17 pregnancies resulting in small-for-gestational-age (SGA) infants, 6 which developed preeclampsia (PE), 1 which developed PE and resulted in an SGA infant, and 37 gestational age-matched controls. The concentration of the PAPP-A2 isoform corresponding to the full-length protein was significantly higher in pregnancies that developed PE (35 ng/mL) compared with those that did not (23 ng/mL; P < .044). In contrast, we found no difference in PAPP-A2 levels between pregnancies that did or did not result in an SGA infant. The upregulation of PAPP-A2 that has previously been observed in PE at term appears to begin early in pregnancy, well before the symptoms develop.

BackgroundAdverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -I and -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. The insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, including sequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and −5 in the placenta are unknown, despite consistent associations between pregnancy complications and the levels of two IGFBP-4 and/or −5 proteases, pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study was to elucidate the effects of IGFBP-4 and −5 on IGF-I and IGF-II in a model of EVT migration. A related objective was to determine the timing and location of IGFBP-4 and −5 expression in the placental villi.MethodsWe used wound healing assays to examine the effects of IGFBP-4 and −5 on the migration of HTR-8/SVneo cells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, −5 and PAPP-A2 was assessed by immunohistochemical staining of first trimester placental sections.Results2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly more than IGF-II. IGFBP-4 and −5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blocked the effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to control levels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistry revealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placental villi as early as 5 weeks of gestational age.ConclusionsIGFBP-4 and −5 show different levels of inhibition on the migration-stimulating effects of IGF-I and IGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and their substrates within placental villi suggests undescribed roles of these molecules in early placental development.Electronic supplementary materialThe online version of this article (doi:10.1186/1477-7827-12-123) contains supplementary material, which is available to authorized users.

Small interfering RNAs (siRNAs) have tremendous potential for the selective inhibition, or ‘silencing’, of genes involved in cancer cell growth and division. This inhibition occurs through a process known as RNA interference (RNAi). Polo-like kinase 1 (PLK1) is a target that has multiple critical roles in cell cycle regulation and cytokinesis. Here we describe the preclinical characterization of TKM-080301, a lipid nanoparticle (LNP) formulation of an siRNA directed against human PLK1 mRNA. Studies were performed to assess the in vitro pharmacologic activity and inherent immune stimulatory potential of various siRNAs. PLK1 siRNA formulated in LNP resulted in potent anti-proliferative activity and gene-specific silencing in many cancer cell lines; and TKM-080301 exhibited strong anti-tumor activity in several xenograft models of human cancer, including tumors implanted intra-hepatically and subcutaneously. RNAi and the intended pharmacologic effects were confirmed in these models by histopathology, to visualize mitotic disruption, and by molecular methods, to confirm the presence of the RNAi-induced PLK1 mRNA cleavage product, the degree of PLK1 silencing relative to housekeeping genes, and the duration of silencing. In vivo, PLK1 silencing persisted for up to 7-10 days after a single administration and, importantly, occurred in the absence of any measurable stimulation of the innate immune system. Finally, unlike most small molecule PLK1 inhibitors, which are highly myelosuppresive, the toxicity profile of TKM-080301 was governed by the distribution profile of the LNP and toxicity was largely restricted to the liver and spleen. Collectively, these studies support the clinical evaluation of TKM-080301 as a new approach to targeting PLK1 in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2829. doi:10.1158/1538-7445.AM2011-2829