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

Abstract

The acute hepatic porphyrias are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks. Factors that induce the expression of hepatic 5-aminolevulinic acid synthase 1 (ALAS1) result in the accumulation of the neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which recent studies indicate are primarily responsible for the acute attacks. Current treatment of these attacks involves i.v. administration of hemin, but a faster-acting, more effective, and safer therapy is needed. Here, we describe preclinical studies of liver-directed small interfering RNAs (siRNAs) targeting Alas1 (Alas1-siRNAs) in a mouse model of acute intermittent porphyria, the most common acute hepatic porphyria. A single i.v. dose of Alas1-siRNA prevented the phenobarbital-induced biochemical acute attacks for approximately 2 wk. Injection of Alas1-siRNA during an induced acute attack significantly decreased plasma ALA and PBG levels within 8 h, more rapidly and effectively than a single hemin infusion. Alas1-siRNA was well tolerated and a therapeutic dose did not cause hepatic heme deficiency. These studies provide proof-of-concept for the clinical development of RNA interference therapy for the prevention and treatment of the acute attacks of the acute hepatic porphyrias.

Keywords: RNAi therapeutics; heme biosynthetic disorders; liver-targeted siRNA.

Fig. 1.

Prophylactic Alas1-siRNA1 prevents PB-induced hepatic Alas1 expression and plasma and urinary ALA and PBG accumulation. AIP mice were i.v. administered Alas1-siRNA1 or Luc-siRNA and then subjected to three daily PB injections starting the following day. Liver, plasma, and urine samples were collected ∼16 h after the last PB injection. Hepatic Alas1 (A) mRNA, (B) protein expression levels, and (C) enzymatic activities were determined. ALA and PBG concentrations were measured in the (D) plasma and (E) urine. Data from wild-type (WT) mice are also shown. In A and C–E, data are presented as mean ± SD (n = 3–8). (B) A representative Western blot image.

Fig. 2.

Preventive effect of a single Alas1-siRNA1 dose is durable and dose-dependent. (A) AIP mice were i.v. administered the Alas1-siRNA1 or Luc-siRNA and then subjected to three daily PB injections at weeks (w) 0, 2, and 4. Plasmas were collected ∼16 h after the last PB injection, and the ALA and PBG levels were determined. Data shown are means ± SDs (n = 4–7). *P < 0.005 versus Luc controls and **P < 0.0005 versus Luc controls, Student t test. (B) AIP mice were i.v. administered 1.0 mg/kg Luc-siRNA or 0.05, 0.1, 0.5, or 1.0 mg/kg of the Alas1-siRNA1 and then challenged with three daily PB injections. Plasma ALA and PBG levels were determined and are shown as mean ± SD (n = 3–6).

Fig. 3.

Alas1-siRNA1 rapidly decreases plasma ALA and PBG concentrations during an ongoing acute attack. AIP mice were biochemically induced with three daily PB injections (110, 130, and 150 mg/kg) and then treated with i.v. ALAS1-siRNA1 (n = 4) or Luc-siRNA (n = 3) ∼12 h after the last PB dose. Plasmas were collected every 4 h, and (A) ALA and (B) PBG levels were monitored. Data represent mean ± SD. *P < 0.05 and **P < 0.00005, Student t test.

Fig. 4.

Alas1-siRNA1 reduces biochemically induced plasma ALA and PBG levels more rapidly and effectively than hemin. Biochemical attacks were induced in the AIP mice by administration of three daily doses of PB (90, 100, and 110 mg/kg) and DDC (20 mg/kg). About 8 h following the last PB/DDC dose, the mice were administered a single i.v. dose of Alas1-siRNA1 (2.0 mg/kg; n = 7), Luc-siRNA (2.0 mg/kg; n = 4), or hemin (4.0 mg/kg; n = 5). Plasma (A) ALA and (B) PBG levels were monitored every 6 h and are presented as mean ± SD. *P < 0.05 vs. hemin, **P < 0.005 vs. hemin, ***P < 0.0005 vs. hemin, and ****P < 0.00005 vs. hemin, Student t test.

Fig. 5.

Alas1-siRNA1 treatment protects against induced neuromotor decline. AIP mice were administered Alas1-siRNA1 (n = 20) or Luc-siRNA (n = 16; both at 2.0 mg/kg) every 1–2 wk and subjected each week to three daily PB (100, 110, and 120 mg/kg/d) and DDC (25 mg/kg) inductions for 4 wk. Neuromotor function was evaluated on a rotarod apparatus rotating at 16 rpm for a maximal time of 180 s. Rotarod testing was performed ∼30 h after the last PB/DDC dose. Data represent mean ± SD. *P = 0.0042, Student t test.

Fig. 6.

Alas1-siRNA1 administration does not alter hepatic heme status or CYP2E1 activity. AIP mice were administered a single 1.0 mg/kg i.v. dose of Alas1-siRNA1 or Luc-siRNA, and the livers were isolated ∼24 h later to assess (A) heme saturation of TDO and (B) CYP2E1 activity. Data are shown as mean ± SD (n = 4–6). *P < 0.01, ANOVA.