DPC characteristics and Clinical chemistry

Abstract 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. Mol Cancer Ther; 17(1); 140–9. ©2017 AACR.

The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.

Title: HIF2α targeted RNAi therapeutic inhibits clear cell renal cell carcinoma Authors and affiliations: So C. Wong, Weijun Cheng, Holly Hamilton, Anthony L. Nicholas, Darren H. Wakefield, Aaron Almeida, Andrei V. Blokhin, Jeffrey Carlson, Zane C. Neal, Vladimir Subbotin, Guofeng Zhang, Julia Hegge, Stephanie Bertin, Vladimir S. Trubetskoy, David B. Rozema, David L. Lewis, Steven B. Kanner 1. Arrowhead Pharmaceuticals Inc., Madison, WI

Introduction: Current anticoagulants effectively prevent thromboembolic (TBE) events, but unmet need remains due to an elevated risk of major bleeding events. Factor XII (F12) autoactivation initia...

Background: Meta-analyses of clinical studies have demonstrated that high levels of lipoprotein(a), known as Lp(a), correlate with residual risk of cardiovascular disease (CVD) while Mendelian genetic studies indicate that Lp(a) is an independent cause of CVD. Lp(a) is a complex lipoprotein particle similar in structure to LDL (consisting of cholesterol, triglycerides, phospholipids and apoB-100), but contains the genetically variable liver-expressed protein apo(a). Lp(a) may contribute to atherosclerosis by targeting oxidized phospholipids to vascular injury sites, promoting both inflammation and accumulation of lipids. Methods: RNA interference (RNAi) based approaches for reducing liver apo(a) expression were developed using both a targeted intravenous platform called Dynamic Polyconjugates (DPC) TM and a subcutaneous (SQ) format. The DPC platform comprises an RNAi trigger conjugated to cholesterol, and a hepatocyte-targeted, membrane active peptide to promote endosomal release of the RNAi trigger. The SQ platform is an RNAi trigger targeted to liver by N-acetyl galactosamine in a proprietary format. RNAi activity was assessed in two different humanized transgenic mice and cynomolgus monkeys. Results: In Lp(a) transgenic mice expressing human apo(a) and apoB-100 genes, treatment with a single DPC dose reduced serum Lp(a) by >98% at nadir and >90% for >5 weeks. A single DPC injection in cynomolgus monkeys resulted in >95% reduction in endogenous Lp(a) particles at 5 weeks post injection, without significant changes in toxicity biomarkers. Treatment with a SQ RNAi trigger reduced serum apo(a) in transgenic mice by >90% at nadir, while that in monkeys reached 64% reduction of Lp(a). Additional modifications to the RNAi trigger improved both depth and duration of knockdown in transgenic mice. Conclusion: Long-duration reductions in circulating apo(a) and Lp(a) have been shown with hepatocyte-directed delivery of apo(a)-specific RNAi triggers in mice and monkeys. This represents an intriguing mechanism to evaluate in humans for reducing residual risk of CVD in LDL-controlled individuals with elevated Lp(a).

Introduction: Mendelian randomization studies and meta-analyses have demonstrated that elevated circulating lipoprotein(a), known as Lp(a), is an independent risk factor for cardiovascular diseases...

A significant medical need exists for improved prophylactic treatment options for Hereditary Angioedema (HAE). Factor 12 (F12) autoactivation in the absence of C1 inhibitor (C1INH) initiates the pathway that leads to bradykinin-mediated edema. We hypothesized that an RNA interference (RNAi) based approach for reducing liver F12 production using our Dynamic Polyconjugate (DPC)™ delivery platform may provide a new prophylactic therapy for HAE. Highly specific and mouse/human/non-human primate (NHP) cross-reactive RNAi triggers were designed in silico and screened for F12 knockdown activity in vitro and in wild type mice. Structure activity relationship (SAR) studies of the most active RNAi triggers identified optimal modifications enabling lead identification. This lead RNAi trigger was further tested for activity and safety in NHPs. Disease-modifying activity in relevant disease-specific mouse models was also explored. Screening of in vitro-active F12 RNAi triggers in wild type mice identified those triggers that exhibited significant and sustained knockdown of serum F12 levels. SAR studies allowed identification of a lead RNAi trigger that demonstrated >97% maximum knockdown after a single 2 mg/kg dose. A multi-dose study in NHPs using 2 mg/kg monthly doses showed >90% sustained knockdown of serum F12 levels without toxicity. NHPs in these studies showed changes in coagulation measurements consistent with F12 deficiency. Studies in mice showed reduced FeCl3-induced thromboembolism consistent with the expected physiological effects of F12 knockdown. Studies in C1INH-deficient mice (HAE model) are currently in progress. Delivery of a potent F12-specific RNAi trigger by DPC™ offers potential for a novel, infrequently-dosed prophylactic treatment for HAE.

Abstract Background: Targeted therapy including VEGF and mTOR pathway inhibitors has dramatically transformed treatment options and outcomes for patients with metastatic clear cell renal cell carcinoma (ccRCC). However, alternate treatments are needed as resistance to these initially promising agents occurs frequently. RNAi interference (RNAi), an innate gene silencing mechanism, has been explored as a new class of therapeutics where conventional treatments are lacking or have failed. The challenge in leveraging this promising approach has been efficient delivery of an RNAi trigger (siRNA) to target tissue. Over 90% of ccRCC tumors express a mutant inactive form of the von Hippel-Landau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong evidence supports the observation that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF-2α), a tumorigenic driver of ccRCC. Methods: We have developed a targeted delivery platform called Dynamic Polyconjugte™ (DPC) as an RNAi-based therapeutic targeting HIF-2α for advanced ccRCC. The ccRCC-specific DPC (ITG-DPC) comprises a membrane active polymer to promote RNAi trigger endosomal release, a ligand that binds to αV-containing integrin receptors expressed on tumor cells, reversible masking to prevent polymer activity before reaching the endosomal compartment, and a potent and specific RNAi trigger to HIF-2α. The modular nature of this delivery platform allows for flexibility to optimize each functional component independently. The ligand-dependent delivery of ITG-DPC was first evaluated in cultured tumor cells and then confirmed in ccRCC tumors established in nude mice using fluorescently-labeled ITG-DPC and confocal microscopy. To validate silencing of HIF-2α as an effective therapeutic approach, an inducible shRNA to HIF-2α was expressed in ccRCC tumors established in mice that significantly silenced HIF-2α gene expression and induced tumor regression. Results: Bi-weekly injection of ITG-DPC into nude mice with established orthotopic A498 ccRCC tumors resulted in >80% knockdown of HIF-2α mRNA and significant tumor growth inhibition. Histological examination of tumor sections showed substantial cell killing and destruction of tumor architecture. Down-regulation of HIF-2α regulated pathways genes including VEGF-A, and the corresponding reduction in tumor-associated CD31 positive neovascularization, corroborated on-target effects of HIF-2α gene silencing. Conclusion: Targeted delivery of a HIF-2α specific RNAi-based-therapeutic has the potential to radically impact the late-stage ccRCC treatment paradigm. Citation Format: So Wong, Weijun Cheng, Darren Wakefield, Aaron Almeida, Andrei Blokhin, Holly Hamilton, Vladimir Subbotin, Julia Hegge, Zane Neal, Guofeng Zhang, David Rozema, David Lewis, Steven Kanner. Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2064.

biopsies were evaluated according to predefined histological scoring system. Staging of fibrosis accounted for bridging fibrous septa normally present in swine livers. Improvement in histological features after FGF21 therapy was defined as a reduction of ≥ 1 point, whereas worsening was defined as an increase of ≥ 1 point compared to pre-therapy biopsy. Insulin sensitivity was assessed with an oral glucose tolerance test. at study (265 vs and triglycerides (142 mg/dL, p not significantly change, significantly lower FGF21 therapy (25 vs 14 μ U/ml, p< 0.05). All liver biopsies prior to initiating FGF21 therapy showed significant fibrosis and extensive hepatocyte ballooning. Following FGF21 therapy, improvement in fibrosis, ballooning, portal and lobu-lar inflammation was noted in 62.5%, 87.5%, 50%, and 25%, respectively. Electron microscopy showed markedly decreased number of secondary lysosomes within hepatocytes and an increased number of lipid-laden Kupffer cells after FGF21 therapy. Except for itching at the injection site, FGF21 was well tolerated. Conclusions: FGF21 therapy results in improve-ments in liver necroinflammation and fibrosis, insulin sensitivity, and post-prandial lipidemia in Ossabaw miniature swine with diet-induced NASH. These observations suggest that FGF21 should be further investigated as a potential treatment for NASH. morphogenetic with the Our novel approaches Acetaminophen (APAP)-induced liver injury is the most fre-quent cause of acute liver failure in the US and many other countries. APAP produces liver cell death by a sequence of events that requires the early formation of a reactive metab-olite, N-acetyl-p-benzoquinone imine (NAPQI), a subsequent phase where adducts of cellular and mitochondrial proteins are formed, and a final phase where these adducts trigger mitochondrial dysfunction and necrotic cell death. Whether hepatocytes can remove the APAP-adducts (APAP-AD) and by which mechanisms are not known. Our recent work has shown that cell survival after APAP overdose is dependent on the autophagy pathway. However, the mechanisms by which autophagy protects against APAP-induced liver injury are not clear. In the present study, using a specific antibody for APAP-AD, we found APAP-AD increased after 1 hr treatment with APAP and reached a maximum around 2 hrs, which started to decline at 6 hrs and reached a relative low level at 24 hrs. These results were also confirmed by HPLC-ED analysis for APAP-AD. Moreover, similar time-dependent changes of APAP-AD were also detected in APAP-treated primary mouse hepatocytes. we found displayed a punctate were with GFP-LC3 positive autophagosomes and LAMP1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, using nycodenz gradient centrifugation approach, we isolated autophagosomes and autolysosomes from control and APAP-treated mouse livers. We found that only the autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD and mitochondria, suggesting autophagy may selectively remove APAP-AD and damaged mitochondria. More importantly, pharmacological inhibition of autophagy by chloroquine increased APAP-AD and damaged mitochondria in APAP-treated mouse livers resulting in increased liver injury. In contrast, pharmacological induction of autophagy by rapamycin decreased APAP-AD and damaged mitochondria and attenuated APAP-induced liver injury. Furthermore, we also found that SQSTM1/p62 (here-after referred to as p62), an autophagy receptor protein, was also recruited to APAP-AD and damaged mitochondria. Ade-novirus-mediated shRNA knockdown of p62 led to increased APAP-AD and necrosis. Collectively, our data indicate for the first time that the level of APAP-AD could be regulated by p62-mediated selective autophagy. Modulating p62-mediated selective autophagy for APAP-AD and damaged mitochondria may be a novel promising approach for treating APAP-induced liver injury. Background: Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is strong evidence that experimental liver fibrosis depends on bacterial translocation. The aim of our study was to define the role of the microbiota in liver fibrosis using conventional and germ-free C57BL/6 mice. Methods and Results: Chronic liver injury was induced by administration of thioac-etamide (TAA) in the drinking water for 21 weeks. Increased liver fibrosis was observed in germ-free mice as compared with conventional mice as assessed by qPCR for collagen α 1(I) and quantification of Sirius red positive area. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells (HSCs), while hepatic mediators of inflammation were not dif-ferent. To rule out the possibility that the microbiota metabo-lizes orally administered TAA prior to absorption, a second model of liver fibrosis was used. Repeated intraperitoneal injections of carbon tetrachloride (CCl 4 ; total of 12 times) confirmed that germ-free mice show more toxin-induced liver injury and fibrosis. In particular, CCl 4 -induced liver injury was much higher in germ-free mice than conventional mice as evidenced by ALT levels. Similarly, a genetic model using Myd88/Trif deficient mice (Myd88 -/- /Trif LPS2/LPS2 ), which lack downstream innate immunity signaling and mimic a germ-free state, had more severe fibrosis and liver injury than wild type mice following CCl 4 injections. Since cells cannot be maintained under germ-free conditions in culture, we isolated parenchymal and non-parenchymal cells from Myd88/Trif deficient mice. Activation of Myd88/Trif PURPOSE: Alpha-1 antitrypsin deficiency (AATD) is an autosomal recessive genetic disorder that causes pulmonary disease in adults and liver disease in children and adults. Wild type alpha-1 antitrypsin (AAT) is a 52 kDa circulating glycopro-tein produced primarily in liver hepatocytes (~90% of total). The E342K substitution, known as the PiZ mutation, results in improper AAT processing and impairs its secretion by hepatocytes. Individuals homozygous for the PiZ allele (PiZZ) have very low serum concentrations of the Z mutant AAT (Z-AAT) and accumulate Z-AAT aggregates in hepatocytes. These aggregates lead to a recurrent cycle of hepatocyte injury and associated fibrosis, which over time leads to liver disease including cirrhosis and hepatocellular carcinoma. We are currently developing a RNAi-based therapeutic to reduce Z-AAT aggregates in liver, which we propose will prevent the progression and development of AATD-associated liver disease in PiZZ individuals. METHODS: A set of RNAi triggers targeting AAT was designed using bioinformatic algorithms and screened for activity in cells in culture. Highly active RNAi triggers were further modified by incorporating unlocked nucleobase analogs (UNAs). The most potent UNAs were tested in a transgenic mouse model expressing the human PiZ mutant allele. In vivo delivery was accomplished by conjugating the UNAs to cholesterol (chol-UNA) to enhance liver uptake and co-injecting each with a hepatocyte targeted and reversibly masked peptide (MLP-(CDM-NAG) designed to enhance endosomal escape. RESULTS: Intravenous injection of candidate chol-UNAs with MLP-(CDM-NAG) led to dose-dependent inhibition of serum Z-AAT of up to 98% in PiZ mice. Reduced levels of serum Z-AAT (>90%) were sustained in PiZ mice given four biweekly injections of the most potent chol-UNA with MLP-(CDM-NAG). In the livers of these mice, greatly reduced levels of soluble and Z-AAT aggregates were observed coincident with improved liver histology. As an indicator of the potency and duration of effect that might be expected in humans, a single co-injection of chol-UNA and MLP-(CDM-NAG) was performed in cynomolgus monkeys. This led to >90% KD of serum AAT with a duration of effect of greater than one month. CONCLUSION: These results demonstrate dramatic and long-lasting reduction of AAT following co-injection of chol-UNA and MLP-(CDM-NAG) in the PiZ mouse and in cynomolgus monkeys. Repeat dosing pre-vented and even reversed accumulation of Z-AAT aggregates in the PiZ mouse model, the cause of liver disease. This RNAi therapeutic holds great promise for the treatment of patients with AATD-associated liver disease.

PURPOSE: Alpha-1 antitrypsin deficiency (AATD) is an autosomal recessive genetic disorder that causes pulmonary disease in adults and liver disease in children and adults.Wild type alpha-1 antitrypsin (AAT) is a 52 kDa circulating glycoprotein produced primarily in liver hepatocytes (~90% of total).The E342K substitution, known as the PiZ mutation, results in improper AAT processing and impairs its secretion by hepatocytes.Individuals homozygous for the PiZ allele (PiZZ) have very low serum concentrations of the Z mutant AAT (Z-AAT) and accumulate Z-AAT aggregates in hepatocytes.These aggregates lead to a recurrent cycle of hepatocyte injury and associated fibrosis, which over time leads to liver disease including cirrhosis and hepatocellular carcinoma.We are currently developing a RNAi-based therapeutic to reduce Z-AAT aggregates in liver, which we propose will prevent the progression and development of AATD-associated liver disease in PiZZ individuals.METHODS: A set of RNAi triggers targeting AAT was designed using bioinformatic algorithms and screened for activity in cells in culture.Highly active RNAi triggers were further modified by incorporating unlocked nucleobase analogs (UNAs).The most potent UNAs were tested in a transgenic mouse model expressing the human PiZ mutant allele.In vivo delivery was accomplished by conjugating the UNAs to cholesterol (chol-UNA) to enhance liver uptake and co-injecting each with a hepatocyte targeted and reversibly masked peptide (MLP-(CDM-NAG) designed to enhance endosomal escape.RESULTS: Intravenous injection of candidate chol-UNAs with MLP-(CDM-NAG) led to dose-dependent inhibition of serum Z-AAT of up to 98% in PiZ mice.Reduced levels of serum Z-AAT (>90%) were sustained in PiZ mice given four biweekly injections of the most potent chol-UNA with MLP-(CDM-NAG).In the livers of these mice, greatly reduced levels of soluble and Z-AAT aggregates were observed coincident with improved liver histology.As an indicator of the potency and duration of effect that might be expected in humans, a single co-injection of chol-UNA and MLP-(CDM-NAG) was performed in cynomolgus monkeys.This led to >90% KD of serum AAT with a duration of effect of greater than one month.CONCLUSION: These results demonstrate dramatic and long-lasting reduction of AAT following co-injection of chol-UNA and MLP-(CDM-NAG) in the PiZ mouse and in cynomolgus monkeys.Repeat dosing prevented and even reversed accumulation of Z-AAT aggregates in the PiZ mouse model, the cause of liver disease.This RNAi therapeutic holds great promise for the treatment of patients with AATD-associated liver disease.