Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key ...parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Abstract
Background: Clear cell renal cell carcinoma (ccRCC) frequently involves the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Loss of VHL functions lead to the accumulation of ...hypoxia-inducible factors (HIFs). HIF2α has been regarded as a key tumorigenic driver of ccRCC and an attractive therapeutic target. Arrowhead has developed a RNA interference therapeutic (HIF2 RNAi) to selectively target and silence HIF2α expression, using a proprietary targeted-RNAi molecule (TRiM™) delivery platform for the treatment of ccRCC. The TRiM™ based Hif2 construct comprises a highly potent RNAi trigger using stabilization chemistries, targeting ligands to facilitate delivery, and structures to enhance pharmacokinetics (PK). The optimization of HIF2 RNAi to enhance the potency and safety profile to maximize the potential clinical success is described.
Methods: Functional optimization of HIF2 RNAi was evaluated in an orthotopic ccRCC tumor xenograft model established with A498 ccRCC cells that stably expresses the reporter gene SEAP (secreted embryonic alkaline phosphatase) as a serum biomarker for monitoring tumor growth. HIF2 RNAi was delivered by intravenous injections. HIF2α gene silencing was evaluated by isolating tumor RNA and measuring relative gene expression by qRT-PCR.
Results: We demonstrate that to achieve deep HIF2α mRNA knockdown (KD), functionalizing HIF2 RNAi with PK enhancement and tumor targeting ligand (TTL) is required. Optimization of the HIF2 RNAi construct enabled a 10-fold improvement in potency. Evaluation of a loading dose regimen improved overall HIF2α mRNA KD compared to a single administration of equal total dosage. Utilizing this strategy, we demonstrated that silencing of HIF2α mRNA (85% KD) resulted in tumor growth inhibition in the A498 xenograft model. Significant improvement in overall survival was also seen in a patient derived xenograft model. Histology evaluation of tumor samples revealed extensive tumor destruction with clusters of apoptotic cells and necrosis. Follow-up studies suggest that loading doses can be administered four hours apart without loss in potency. This allows dosing to be completed in one day and may be more acceptable in clinical settings. The maximum HIF2α mRNA KD after a single dose of HIF2 RNAi was achieved about 7 days after dosing and sustained for about one week in the xenograft model. This suggests that dosing can likely be less frequent in clinical settings. An exploratory toxicity study in rats predicts a wide safety margin.
Conclusions: We demonstrate that the TRiM™ delivery platform can be utilized to deliver a RNAi therapeutic selectively targeting HIF2α for the treatment of ccRCC. This represents a novel therapeutic approach either as a monotherapy or in combination with other therapies in seeking better tolerated and/or more effective treatment for ccRCC.
Citation Format: So C. Wong, Anthony Nicholas, Jeff Carlson, Dongxu Shu, Che Liu, Rui Chu, Amanda Frankiewicz, Holly Hamilton, Casi Schienebeck, Aaron Andersen, Matthew Fowler-Watters, Stephanie Bertin, Xiaokai Li, Bo Chen, Josh Schumacher, Julia Hegge, Bruce Given, Zhen Li. Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4775.
Abstract Objective This study examined a risk-factor–based assignment to either a subspecialist or a general gynaecologist for the management of women with endometrial cancer. Method At diagnosis, ...all women with a diagnosis of endometrial cancer in four community hospitals were referred to a central cancer centre and stratified into low- and high-risk groups. Risk stratification was based primarily on central pathology review, with low-risk disease defined as grade 1, clinical stage 1. Women with low-risk disease were triaged back to the referring gynaecologist for surgery. Women with high-risk disease were managed at the cancer centre. The main outcome measures included risk status and pathology review, treatment and treatment location, and acceptability to patients and gynaecologists. Results Seventy-three women participated in this pilot study between November 2009 and 2010. Risk stratification was performed in all women: 37 were classified as high risk and 36 as low risk. Ninety-seven percent of women with high-risk disease were managed at the cancer centre, and 83% of these women underwent surgical staging compared with 8% for women with low-risk disease. This approach was acceptable to both patients and gynaecologists. Conclusion This structured pattern of care for women with endometrial cancer resulted in a shift in management, with more women managed in accordance with oncologic guidelines, meaning that women at high risk for metastases had a lymphadenectomy performed.
Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most ...patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model.
.
IntroductionHuman genetic analysis identified that individuals with loss-of-function mutations in angiopoietin-like protein 3 (ANGPLT3) have very low plasma levels of triglycerides (TGs) and ...low-density lipoprotein (LDL-C), and a reduced risk of cardiovascular disease. An RNA interference (RNAi) based therapy using Arrowhead Pharmaceuticals’ hepatocyte targeting delivery TRiM™ platform to reduce ANGPLT3 production in the liver by gene silencing may be an effective approach to treat hyperlipidemia.MethodsHighly specific ANGPTL3 RNAi triggers cross-reactive to human, rodent and non-human primate-(NHPs) transcripts were identified and studied for mRNA knockdown activity. Lead optimization studies in mice identified a lead RNAi trigger that was then tested in NHPs for potency and safety. In addition, lipid lowering activities were evaluated in dyslipidemic mouse and NHP models.ResultsStudies in mice identified a lead trigger that reduces serum ANGPTL3 proteins by 80% after a single subcutaneous (SC) 0.5 mg/kg dose in normal chow-fed mice or a 4 mg/kg dose in lean cynomolgus monkeys. A dose response study in mice determined that maximum efficacious dose was between 3 - 5 mg/kg. To better evaluate the lipid lowering activities, two monthly doses of 3 mg/kg of the lead trigger was administered to three dyslipidemic models of LDL receptor (LDLr) knockout mice, diet-induced obese (DIO) mice, and leptin receptor deficient (db/db) mice. In all three models, maximum serum ANGPLT3 was reduced at least 98%, and reductions in TGs and LDL-C were also observed. Consistent with ANGPTL3’s role in lowering LDL-C independent of LDLr, the LDLr mice showed LDL-C reductions of 50%. In a study using high fat-diet fed dyslipidemic rhesus monkeys, close to 90% reduction in serum ANGPTL3 was achieved after a single SC dose of 4 mg/kg. Explorative safety studies in rat and NHP showed no remarkable concerns.ConclusionsOur results support the potential of an ANGPLT3 targeted RNAi therapeutic as a treatment for hyperlipidemia.
Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III ...shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.
Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine ...how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.
One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and ...associated with cellular membranes that are flat, positively or negatively curved. Using
and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.
PDF
