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Rnai-Mediated Silencing of Hepatic Alas1 Effectively Prevents and Treats the Induced Acute Attacks in Acute Intermittent Porphyria Mice

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Rnai-Mediated Silencing of Hepatic Alas1 Effectively Prevents and Treats the Induced Acute Attacks in Acute Intermittent Porphyria Mice RNAi-mediated silencing of hepatic Alas1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice Makiko Yasudaa, Lin Gana, Brenden Chena, Senkottuvelan Kadirvela, Chunli Yua, John D. Phillipsb, Maria I. Newc,1, Abigail Liebowd, Kevin Fitzgeraldd, William Querbesd, and Robert J. Desnicka,1 aDepartment of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bHematology Division, University of Utah School of Medicine, Salt Lake City, UT 84132; cDepartment of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and dAlnylam Pharmaceuticals, Cambridge, MA 02142 Contributed by Maria I. New, April 16, 2014 (sent for review March 10, 2014) The acute hepatic porphyrias are inherited disorders of heme Currently, the standard therapy for the acute neurovisceral biosynthesis characterized by life-threatening acute neurovisceral attacks is the i.v. administration of hemin, which provides ex- attacks. Factors that induce the expression of hepatic 5-aminolevulinic ogenous heme for the negative feedback inhibition of ALAS1, acid synthase 1 (ALAS1) result in the accumulation of the neuro- thereby decreasing ALA and PBG production (11–13). Although toxic porphyrin precursors 5-aminolevulinic acid (ALA) and por- patients generally respond well, its effect is slow, typically re- phobilinogen (PBG), which recent studies indicate are primarily quiring three to four daily infusions to normalize the elevated responsible for the acute attacks. Current treatment of these plasma and urinary ALA and PBG concentrations (14). Patients attacks involves i.v. administration of hemin, but a faster-acting, who experience recurring attacks are treated prophylactically more effective, and safer therapy is needed. Here, we describe pre- with weekly to monthly hemin infusions, which are associated clinical studies of liver-directed small interfering RNAs (siRNAs) tar- with side effects such as phlebitis and iron overload (15). Thus, geting Alas1 (Alas1-siRNAs) in a mouse model of acute intermittent an alternative therapeutic approach for the acute hepatic por- porphyria, the most common acute hepatic porphyria. A single i.v. phyrias that is more effective, faster-acting, and safer is desirable. dose of Alas1-siRNA prevented the phenobarbital-induced bio- Previously, the pathogenic mechanism of the acute neuro- visceral attacks was unclear, particularly whether the attacks chemical acute attacks for approximately 2 wk. Injection of Alas1- were due to the neurotoxic effects of the accumulated ALA siRNA during an induced acute attack significantly decreased and/or PBG or to the heme deficiency in neuronal tissues (16). plasma ALA and PBG levels within 8 h, more rapidly and effectively However, nonporphyric liver transplant recipients who received than a single hemin infusion. Alas1-siRNA was well tolerated and livers from AIP patients (i.e., “domino” liver transplants) de- a therapeutic dose did not cause hepatic heme deficiency. These veloped acute porphyric attacks despite their presumably normal studies provide proof-of-concept for the clinical development of neuronal heme levels, which demonstrated that the primary RNA interference therapy for the prevention and treatment of the pathogenic mechanism is not neuronal heme deficiency but acute attacks of the acute hepatic porphyrias. rather the effects of the neurotoxic hepatic metabolites ALA MEDICAL SCIENCES and/or PBG (17). Consistent with this finding, orthotopic liver RNAi therapeutics | liver-targeted siRNA | heme biosynthetic disorders transplantation in AIP patients and a VP patient with recurrent he acute hepatic porphyrias are caused by the deficient ac- Significance Ttivities of specific enzymes in the heme biosynthetic path- — way and include three autosomal dominant diseases acute There is an unmet need for a more effective, faster-acting, and intermittent porphyria (AIP), hereditary coproporphyria (HCP), safer therapy for the life-threatening acute neurovisceral attacks — and variegate porphyria (VP) and the autosomal recessive that occur in the acute hepatic porphyrias. Recent studies indicate 5-aminolevulinic acid dehydratase deficiency porphyria (ADP) (1). that the acute attacks are primarily caused by the neurotoxic These inherited metabolic disorders are characterized by life- porphyrin precursors 5-aminolevulinic acid and porphobilinogen, threatening acute neurovisceral attacks that include severe ab- which accumulate as a consequence of the markedly induced dominal pain, hypertension, tachycardia, constipation, motor expression of hepatic 5-aminolevulinic acid synthase 1 (ALAS1). weakness, seizures, and paralysis (1). Various factors, including Here, we demonstrate that liver-targeted small interfering RNAs cytochrome P450 (CYP)-inducing drugs, dieting, and hormonal specific for Alas1 are highly effective in preventing and treating changes precipitate the acute attacks by increasing the expres- the biochemically induced acute attacks in a mouse model of sion of hepatic 5-aminolevulinic acid synthase (ALAS1), the first acute intermittent porphyria, the most common acute hepatic – and rate-limiting enzyme of the heme biosynthetic pathway (2 porphyria. These preclinical studies provide proof-of-concept for 6). When hepatic ALAS1 is induced, the respective enzyme the clinical development of RNAi-mediated therapy for the acute deficiencies create a metabolic bottleneck, thereby decreasing porphyric attacks. hepatic heme production and depleting the “free” heme pool. This leads to the loss of the negative feedback inhibition of heme Author contributions: M.Y., K.F., W.Q., and R.J.D. designed research; M.Y., L.G., B.C., S.K., on ALAS1 (7–9) and, consequently, further up-regulation of C.Y., J.D.P., A.L., and W.Q. performed research; M.Y., M.I.N., W.Q., and R.J.D. analyzed hepatic ALAS1 expression. As hydroxymethylbilane synthase data; and M.Y., M.I.N., W.Q., and R.J.D. wrote the paper. (HMBS)—the third enzyme in the heme biosynthetic pathway—is Conflict of interest statement: A.L., K.F., and W.Q. are employees and shareholders of Alnylam Pharmaceuticals; and M.Y. and R.J.D. are consultants to Alnylam Pharmaceut- less abundant in comparison with the other heme biosynthetic icals. The authors have filed a patent covering RNAi therapies for the treatment enzymes (10), it becomes rate-limiting when ALAS1 is markedly of porphyrias. increased. As a result, the neurotoxic porphyrin precursors Freely available online through the PNAS open access option. 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) are 1To whom correspondence may be addressed. E-mail: [email protected] or robert. overproduced in the liver and are markedly accumulated in the [email protected]. plasma and urine during the acute attacks (only ALA accumulates This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in ADP). 1073/pnas.1406228111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1406228111 PNAS | May 27, 2014 | vol. 111 | no. 21 | 7777–7782 Downloaded by guest on September 26, 2021 and incapacitating attacks resulted in the rapid normalization of ALA and PBG levels and the abrupt remission of attacks (18– 20). Thus, the liver is the primary site of pathology in the acute hepatic porphyrias, and therapies designed to inhibit the induced overexpression of ALAS1 and the resulting elevation of ALA and PBG would prevent or treat the acute attacks in all four acute hepatic porphyrias. Here, we report preclinical studies of a liver-directed RNA in- terference (RNAi) therapy using an i.v.-administered small in- terfering RNA (siRNA) specific for the Alas1 mRNA (designated Alas1-siRNA). To identify a potent Alas1-siRNA, a panel of siRNAs targeting Alas1 was initially screened for their ability to inhibit Alas1 mRNA expression in cultured hepatic cells. The most active compounds were formulated into lipid nanoparticles (LNPs) for efficient hepatic delivery (21–23) and evaluated for their ability to down-regulate hepatic Alas1 mRNA expression in wild-type mice. The selected Alas1-siRNA was then evaluated in the mouse model for AIP for its effectiveness to prevent or treat an induced acute attack. These mice have ∼30% of wild-type HMBS activity and slightly elevated baseline plasma and urinary ALA and PBG levels (24). When phenobarbital (PB), a potent Fig. 1. Prophylactic Alas1-siRNA1 prevents PB-induced hepatic Alas1 expres- inducer of ALAS1 expression, is administered, ALA and PBG are sion and plasma and urinary ALA and PBG accumulation. AIP mice were i.v. markedly increased (20- to 30-fold and 40- to 90-fold in plasma, administered Alas1-siRNA1 or Luc-siRNA and then subjected to three daily PB respectively), mimicking the biochemical pathology of a human injections starting the following day. Liver, plasma, and urine samples were acute attack (1, 24). Prophylactic administration of Alas1-siRNA collected ∼16 h after the last PB injection. Hepatic Alas1 (A)mRNA,(B)protein in the AIP mice completely prevented the PB-induced accumu- expression levels, and (C) enzymatic activities were determined. ALA and PBG lation of plasma and urinary ALA and PBG, whereas infusion of concentrations were measured in the (D) plasma and (E) urine. Data from wild- Alas1-siRNA after induction of an acute attack rapidly and ef- type (WT) mice are also shown. In A and C–E, data are presented as mean ± SD fectively decreased the markedly elevated plasma ALA and PBG. (n = 3–8). (B) A representative Western blot image. Results Alas1 A–C Selected Alas1-siRNA Robustly Silenced Endogenous Alas1 mRNA hepatic expression levels (Fig. 1 ). Sixteen hours after Expression in Wild-Type Mice. To identify a potent siRNA se- the last PB injection, mean plasma ALA and PBG concen- quence, 45 Alas1-targeted siRNAs were evaluated for their ability trations in the Luc-siRNA controls were elevated ∼21- and 91- to repress Alas1 mRNA expression in a murine BNL-C12 hepa- fold from baseline levels of 0.46 ± 0.16 and 0.19 ± 0.16 μmol/L to tocyte cell line. The two most active siRNAs were individually 9.7 ± 3.6 and 17 ± 4.2 μmol/L, respectively (Fig. 1D). Urinary formulated into LNPs and i.v.
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