MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.National Institutes of Health (U.S.) (GM0077465)National Institutes of Health (U.S.) (DK080261

Manufacture of therapeutic oligonucleotides: Development of new reagents and processes

2297-2304The optimization of synthesis, purification process parameters and development of new reagents have enabled successful large-scale manufacture of several therapeutic grade oligonucleotides at Avecia. The development of new reagents and processes are described in this communication

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders

RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver

BACKGROUND and AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver

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

Allele-Selective Inhibition of Mutant Atrophin‑1 Expression by Duplex and Single-Stranded RNAs

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (<i>ATN-1</i>) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant <i>ATN-1</i> were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (<i>ATXN-3</i>) and huntingtin (<i>HTT</i>). These data demonstrate that the expanded trinucleotide repeat within <i>ATN-1</i> mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes

MICU2 is paralogous to MICU1 and localizes to mitochondria.

<p>A. MICU1, MICU2 and MICU3 share a common ancestor and are present in multiple vertebrate species. B. RNA expression analysis of MICU1, MICU2, MICU3 and MCU across 21 mouse tissues. For each tissue, the dots represent individual replicate measures and the bars represent mean values. C. MICU2 has two evolutionarily conserved EF hands. D. Representative confocal images of HeLa cells cotransfected with MICU2-GFP and Mito-HcRed1.</p

MICU1 and MICU2 can be silenced <i>in vivo</i> in mouse liver using siRNA technology.

<p>A. <i>In vitro</i> dose-response curves of selected duplexes targeting MICU1 and MICU2. B. Relative expression of MICU1 and MICU2 mRNA after 6 weekly injections normalized to siLUC mice. C. Representative oxygen consumption traces measured in isolated mitochondria from siLUC (top) and siMICU1+2 (bottom) mice. Arrows denote addition of mitochondria, glutamate and malate (G/M), ADP and uncoupler (carbonyl cyanide m-chlorophenylhydrazone, CCCP). Respiratory control ratios (RCR) and ADP∶O ratios (P∶O) were calculated from experiments performed on three separate mice per group. D. Representative mitochondrial membrane potential traces measured in isolated mitochondria from siLUC (top) and siMICU1+2 (bottom) mice using tetramethyl rhodamine methyl ester (TMRM). E. Respiratory control ratios (RCR) and ADP∶O ratios (P∶O) were comparable among all treatment groups.</p

MICU1 and MICU2 stabilize each other's expression and interact with MCU.

<p>A. Whole cell lysates from HEK293T cells stably expressing a control shRNA (shGFP and shLACZ) or a shRNA targeting MICU1 (shMICU1_a and shMICU1_b) or MICU2 (shMICU2_a) were analyzed using qPCR and western blot. The relative mRNA is reported using β-actin as an endogenous control and normalized to shGFP for each target. Whole cell lysates were blotted with anti-MICU1, anti-MICU2 and control anti-ATP5A. B. Whole cell lysates from HEK293T cells stably expressing FLAG-GFP or FLAG-MICU1 were lysed and blotted with anti-MICU2, anti-FLAG and control anti-ATP5A. C–D. Mitochondria isolated from HEK293T cells stably expressing MCU-FLAG (C) or FLAG-MICU1 (D) were solubilized with 0.2% DDM and subjected to anti-FLAG immunoprecipitation. Immunoprecipitates and lysate were blotted with anti-FLAG, anti-MICU1, anti-MICU2 and control anti-ATP5B and anti-SDHB.</p

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes