RNAi-mediated silencing of hepatic
            <i>Alas1</i>
            effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice

Yasuda, Makiko; Gan, Lin; Chen, Brenden; Kadirvel, Senkottuvelan; Yu, Chunli; Phillips, John D.; New, Maria I.; Liebow, Abigail; Fitzgerald, Kevin; Querbes, William; Desnick, Robert J.

doi:10.1073/pnas.1406228111

Cited by 109 publications

(103 citation statements)

References 38 publications

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“…These findings suggest that the hepatic heme deficiency in AIP is confined to depletion of the "free" heme pool, leading to greater induction of ALAS1 and accumulation of the putatively neurotoxic ALA and/or PBG. Importantly, these findings validate the use of a liver-directed RNA interference (RNAi) therapeutic targeting hepatic ALAS1 mRNA for the prevention and treatment of acute attacks, as shown in recent preclinical studies (9).…”

Section: Discussionsupporting

confidence: 69%

“…The supernatant was placed on a VWR centrifugal filter and centrifuged at 10,000g for 10 min at 4°C. Hepatic, plasma and urinary ALA and PBG concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as previously described (9,10).…”

Section: Porphyrin Precursor and Porphyrin Isomer Quantitationmentioning

confidence: 99%

“…ALAS1 and HMB synthase activities were determined in homogenized livers as previously described (9,11). Uroporphyrin fluorescence was measured in a Foci Farrand Spectrofluorometer System 3 (Farrand Optical Components and Instruments).…”

Section: Alas1 and Hmb Synthase Enzyme Activity Assaysmentioning

confidence: 99%

“…RNA isolation, reverse transcription and real-time polymerase chain reaction (PCR) were performed as previously described (9), by using predesigned Taqman® assays (Life Technologies) for ALAS1 (assay ID: Hs00963534_m1), HO-1 (Hs01110251_m1), interleukin (IL)-1β (IL-1β; Hs01555410_m1), IL-6 (IL-6; Hs00985639_m1), tumor necrosis factor α (TNFα; Hs01113624_g1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02758991_g1). Relative ALAS1, HO-1, IL-1β, IL-6 and TNFα transcript levels were determined by the comparative C t method using GAPDH as an internal control.…”

Section: Rna Extraction and Real-time Pcr Quantificationmentioning

confidence: 99%

“…ALAS1 Western blot analysis was performed as previously described (9), with the following modifications. Liver samples were homogenized in chilled lysis buffer (20 mmol/L HEPES, 150 mmol/L sodium chloride, pH 7.4, 0.2% IGEPAL, 0.5% Triton X-100 and protease inhibitor cocktail [Sigma-Aldrich]), and protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent Concentrate kit.…”

Section: Western Blot Analysesmentioning

confidence: 99%

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Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver

Yasuda

Erwin

Liu

et al. 2015

Mol Med

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Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal-dominant disorder due to the half-normal activity of the heme biosynthetic enzyme, hydroxymethylbilane (HMB) synthase (1). Symptomatic heterozygotes, most (~90%) of which are women, experience episodic life-threatening acute neurovisceral attacks that typically begin with severe abdominal pain and may include hypertension, tachycardia, constipation, motor weakness and seizures. These attacks are precipitated by certain drugs, dieting and hormonal factors that increase the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1) (1). When hepatic ALAS1 is induced, the half-normal activity of HMB synthase becomes ratelimiting, leading to decreased heme biosynthesis and depletion of the hepatic "free" heme pool. Depletion of the"free" heme pool leads to further induction of Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3-and 5-fold, and ALA and PBG concentrations were increased ~3-and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

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Section: Discussionsupporting

confidence: 69%

Section: Porphyrin Precursor and Porphyrin Isomer Quantitationmentioning

confidence: 99%

Section: Alas1 and Hmb Synthase Enzyme Activity Assaysmentioning

confidence: 99%

Section: Rna Extraction and Real-time Pcr Quantificationmentioning

confidence: 99%

Section: Western Blot Analysesmentioning

confidence: 99%

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Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver

Yasuda

Erwin

Liu

et al. 2015

Mol Med

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Charge‐Conversion Strategies for Nucleic Acid Delivery

Dutta

Das

Medeiros

et al. 2021

Adv Funct Materials

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Nucleic acids are considered as one of the most potent therapeutic modalities, as their roles go beyond storing genetic information and chemical energy or as signal transducers. Attenuation or expression of desired genes through nucleic acids have profound implications in gene therapy, gene editing, and even in vaccine development. Although nucleic acid therapeutics bring in overwhelming possibilities toward the development of molecular medicines, there are significant loopholes in their effective clinical translation. One of the major pitfalls lies in the traditional design concepts of nucleic acid drug carriers, namely, cationic charge induced cytotoxicity. Targeting this bottleneck, several innovative carrier designs have been proposed accommodating charge‐conversion approaches, whereby built‐in functionalities convert from cationic to neutral or anionic, or even from anionic to cationic enabling the carrier to overcome several critical barriers for therapeutics delivery, such as serum deactivation, instability in circulation, low transfection, and poor endosomal escape. This review will critically analyze various molecular designs of charge‐converting nanocarriers in a classified approach for the successful delivery of nucleic acids. Accompanied by the narrative on recent clinical nucleic acid candidates, the review concludes with a discussion on the pitfalls and scope of these emerging approaches.

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Biomaterials in siRNA Delivery: A Comprehensive Review

Zhang

2016

Adv Healthcare Materials

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With the dearth of effective treatment options for prominent diseases including Ebola and cancer, RNA interference (RNAi), a sequence-specific mechanism for genetic regulation that can silence nearly any gene, holds the promise of unlimited potential in treating illness ever since its discovery in 1999. Given the large size, unstable tertiary structure in physiological conditions and negative charge of small interfering RNAs (siRNAs), the development of safe and effective delivery vehicles is of critical importance in order to drive the widespread use of RNAi therapeutics into clinical settings. Immense amounts of time and billions of dollars have been devoted into the design of novel and diverse delivery strategies, and there are a handful of delivery systems that have been successfully translated into clinic. This review provides an introduction to the in vivo barriers that need to be addressed by siRNA delivery systems. We also discuss the progress up to the most effective and clinically advanced siRNA delivery systems including liposomal, polymeric and siRNA conjugate delivery systems, as well as their design to overcome the challenges.

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RNAi-mediated silencing of hepatic Alas1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice

Cited by 109 publications

References 38 publications

Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver

Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver

Charge‐Conversion Strategies for Nucleic Acid Delivery

Biomaterials in siRNA Delivery: A Comprehensive Review

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