Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration.

Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

Progesterone receptor membrane component 1 (PGRMC1) binds and stabilizes cytochromes P450 through a heme-independent mechanism.

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.

Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.

Thrombin generation in plasma of patients with haemophilia A and B with inhibitors: Effects of bypassing agents and antithrombin reduction.

Antithrombin (AT) reduction has been shown to improve thrombin generation (TG) in haemophilia with or without inhibitors. As treatment with bypassing agents (BPAs) may be required in patients with breakthrough bleeding while receiving AT-lowering therapy, we assessed TG in platelet-poor plasma samples from haemophilia patients in the presence of BPA (recombinant activated factor VII [rFVIIa; 1.25 or 2.5 µg mL-1] or activated prothrombin complex concentrate [aPCC; 0.5 or 1 U mL-1]) and AT reduction (anti-AT antibody). Mean ± SEM baseline peak thrombin height was 19.9 ± 4.3 nM in plasma from haemophilia patients (n = 12) and 230.5 ± 9.8 nM in healthy males (n = 24). Reduced AT improved mean peak thrombin height in haemophilia patient plasma to 75.4 ± 17.4 nM. Spiking of 90% AT-reduced haemophilia patient plasma with 2.5 µg mL-1 rFVIIa or 1 U mL-1 aPCC increased the mean peak thrombin height to 82.5 ± 12 nM and 134.8 ± 18.7 nM, respectively. Peak thrombin levels did not exceed the range for healthy volunteers when plasma samples from haemophilia patients with in vitro AT reduction were treated with BPAs, suggesting the potential use of BPAs in conjunction with AT reduction. Further clinical investigations are needed to confirm the safety of this approach.

Endothelial TGF-ß signalling drives vascular inflammation and atherosclerosis.

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial TGFß signaling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGFß signaling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These pro-inflammatory effects of endothelial TGFß signaling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type specific therapeutic intervention aimed at control of this disease.

Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates.

Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.

The Polyploid State Plays a Tumor-Suppressive Role in the Liver.

Most cells in the liver are polyploid, but the functional role of polyploidy is unknown. Polyploidization occurs through cytokinesis failure and endoreduplication around the time of weaning. To interrogate polyploidy while avoiding irreversible manipulations of essential cell-cycle genes, we developed orthogonal mouse models to transiently and potently alter liver ploidy. Premature weaning, as well as knockdown of E2f8 or Anln, allowed us to toggle between diploid and polyploid states. While there was no detectable impact of ploidy alterations on liver function, metabolism, or regeneration, mice with more polyploid hepatocytes suppressed tumorigenesis and mice with more diploid hepatocytes accelerated tumorigenesis in mutagen- and high-fat-induced models. Mechanistically, the diploid state was more susceptible to Cas9-mediated tumor-suppressor loss but was similarly susceptible to MYC oncogene activation, indicating that polyploidy differentially protected the liver from distinct genomic aberrations. This suggests that polyploidy evolved in part to prevent malignant outcomes of liver injury.

Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.

Knockdown of Anillin Actin Binding Protein Blocks Cytokinesis in Hepatocytes and Reduces Liver Tumor Development in Mice Without Affecting Regeneration.

BACKGROUND & AIMS: Cytokinesis can fail during normal postnatal liver development, leading to polyploid hepatocytes. We investigated whether inhibiting cytokinesis in the liver slows tumor growth without compromising the health of normal hepatocytes. We inhibited cytokinesis in cancer cells by knocking down ANLN, a cytoskeletal scaffolding protein that regulates cytokinesis and might promote tumorigenesis, in mice with liver disease. METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas, Oncomine, PrognoScan, and a hepatocellular carcinoma (HCC) tissue microarray. We knocked down ANLN with small interfering RNAs (siRNAs) in H2.35 liver cells and performed image analyses of cells undergoing cytokinesis. siRNAs were delivered to LAP-MYC mice, which develop hepatoblastoma, using lipid nanoparticles. H2.35 cells with knockdown of ANLN or control cells were injected into FRG mice, which develop chronic liver damage, and tumor growth was monitored. We also developed mice with inducible expression of transgenes encoding small hairpin RNAs (shRNAs) against Anln messenger RNA and studied liver tumorigenesis after administration of diethylnitrosamine and carbon tetrachloride. siRNAs against Anln messenger RNA were conjugated to N-acetylgalactosamine to reduce toxicity and increase hepatocyte tropism; their effects were studied in mouse models of liver cancer and regeneration. RESULTS: Levels of ANLN messenger RNA were increased in human HCC tissues compared to non-tumor liver tissues. siRNA knockdown of ANLN blocked cytokinesis in H2.35 liver cells. Administration of siRNA against ANLN increased survival times of LAP-MYC mice, compared to mice given a control siRNA. H2.35 liver cells with shRNA knockdown of ANLN formed tumors more slowly in FRG mice than control H2.35 cells. Mice with inducible expression of shRNAs against Anln mRNA developed fewer liver tumors after administration of diethylnitrosamine and carbon tetrachloride than control mice. Knockdown of ANLN did not affect liver regeneration after acute and chronic liver injuries. CONCLUSIONS: Knockdown of ANLN in liver cells blocks cytokinesis and inhibits development of liver tumors in mice. Agents that inhibit ANLN in the liver might be effective for prevention or treatment of HCC.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Methods to Identify and Characterize siRNAs Targeting Serpin A1 In Vitro and In Vivo Using RNA Interference.

RNAi is a powerful tool that can be used to probe gene function as well as for therapeutic intervention. We describe a workflow and methods to identify screen and select potent and specific siRNAs in vitro and in vivo using qPCR-based methods as well as an AAT activity assay. We apply these techniques to a set of siRNAs targeting rat AAT, and use this set to exemplify the cell-based and in vivo data that can be generated using these methods.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Specific hepatic delivery of procollagen α1(I) small interfering RNA in lipid-like nanoparticles resolves liver fibrosis.

UNLABELLED: Fibrosis accompanies the wound-healing response to chronic liver injury and is characterized by excessive hepatic collagen accumulation dominated by collagen type I. Fibrosis often progresses to cirrhosis. Here we present in vivo evidence of an up to 90% suppression of procollagen α1(I) expression, a reduction of septa formation, and a 40%-60% decrease of collagen deposition in mice with progressive and advanced liver fibrosis that received cationic lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene. After intravenous injection, up to 90% of lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene were retained in the liver of fibrotic mice and accumulated in nonparenchymal more than parenchymal cells for prolonged periods, significantly ameliorating progression and accelerating regression of fibrosis. CONCLUSION: Our lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene specifically reduce total hepatic collagen content without detectable side effects, potentially qualifying as a therapy for fibrotic liver diseases.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

The roles of individual mammalian argonautes in RNA interference in vivo.

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.

Tissue-specific gene silencing monitored in circulating RNA.

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies and gene therapy. Here we show that mRNAs encoding tissue-specific gene transcripts can be detected in biological fluids and that RNAi-mediated target gene silencing in the liver and brain results in quantitative reductions in serum and cerebrospinal fluid mRNA levels, respectively. Further, administration of an anti-miRNA oligonucleotide resulted in decreased levels of the miRNA in circulation. Moreover, ectopic expression of an adenoviral transgene in the liver was quantified based on measurement of serum mRNA levels. This noninvasive method for monitoring tissue-specific RNA modulation could greatly advance the clinical development of RNA-based therapeutics.

Safety and efficacy of RNAi therapy for transthyretin amyloidosis.

BACKGROUND: Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS: We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS: Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS: ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).