mRNA-based therapy in a rabbit model of variegate porphyria offers new insights into the pathogenesis of acute attacks

Abstract

Variegate porphyria (VP) results from haploinsufficiency of protoporphyrinogen oxidase (PPOX), the seventh enzyme in the heme synthesis pathway. There is no VP model that recapitulates the clinical manifestations of acute attacks. Combined administrations of 2-allyl-2-isopropylacetamide and rifampicin in rabbits halved hepatic PPOX activity, resulting in increased accumulation of a potentially neurotoxic heme precursor, lipid peroxidation, inflammation, and hepatocyte cytoplasmic stress. Rabbits also showed hypertension, motor impairment, reduced activity of critical mitochondrial hemoprotein functions, and altered glucose homeostasis. Hemin treatment only resulted in a slight drop in heme precursor accumulation but further increased hepatic heme catabolism, inflammation, and cytoplasmic stress. Hemin replenishment did protect against hypertension, but it failed to restore action potentials in the sciatic nerve or glucose homeostasis. Systemic porphobilinogen deaminase (PBGD) mRNA administration increased hepatic PBGD activity, the third enzyme of the pathway, and rapidly normalized serum and urine porphyrin precursor levels. All features studied were improved, including those related to critical hemoprotein functions. In conclusion, the VP model recapitulates the biochemical characteristics and some clinical manifestations associated with severe acute attacks in humans. Systemic PBGD mRNA provided successful protection against the acute attack, indicating that PBGD, and not PPOX, was the critical enzyme for hepatic heme synthesis in VP rabbits.

Keywords: characterization of variegate porphyria rabbits; hepatic heme synthesis; lipid nanoparticles; mRNA delivery; pharmacological model of variegate porphyria; rare metabolic disease.

Graphical abstract

Figure 1

Biochemical characterization of the variegate porphyria model in rabbits and the effects of common and emerging therapies for acute attacks, hemin, and hPBGD mRNA, respectively Eleven doses of AIA (350 mg/kg, s.c.) and twelve doses of rifampicin (200 mg/kg, i.p.) were administered to 13 female New Zealand rabbits during 20 days. Of these, hemin (8 mg/kg) was i.p. injected into four animals on days 15–18 (brown arrows) and hPBGD mRNA (0.5 mg/kg) was i.v. administered on day 16 (blue arrow) in another three rabbits. Seven un-injected rabbits were used as a reference group. (A) PPOX activity and the expression of the two regulatory enzymes of heme metabolism and catabolism, (B) Alas1 and (C) Ho-1, respectively, were measured in liver samples 16 h after the last rifampicin administration. Rabbits were housed in individual cages, and 24-h urine samples were collected during challenges. (D) Total urinary loss of heme precursors on day 20 reported as nmol ALA per 24 h (two ALA molecules are required to synthesize a PBG monopyrrole, and eight ALA molecules are required to synthesize one porphyrin). (E) Urinary PBG excretion over time. (F) Quantification of the total peak area of the urinary PBG and ALA excretion between days 17 and 20. (G) Urinary excretion of individual porphyrins and (H) total plasma porphyrin levels on day 20. (I) Hepatic PBGD activity measured at sacrifice. Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 against the non-injected group. VP, variegate porphyria; AIA, allyl-2-isopropylacetamide; rif, rifampicin; PPOX, protoporphyrinogen oxidase; ALA, δ-aminolevulinic acid; ALAS1, ALA-synthase 1; PBG, porphobilinogen.

Figure 2

Liver status and function in rabbits challenged with AIA and rifampicin (n = 6) and the effects of the administration of hemin (n = 4) and hPBGD mRNA (n = 3) Serum (A) ALP and (B) ALT levels from cardiac-puncture blood samples taken at sacrifice (day 20). (C) Cumulative activity of the mitochondrial respiratory complexes I–IV. (D) Mitochondrial complex IV content as quantified by western blot from liver samples. (E) Total heme and (F) mitochondrial heme a content measured in liver samples. Control group corresponds to non-injected rabbits (n = 6). Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus the non-injected group.

Figure 3

Protective efficacy of a single i.v. administration of hPBGD mRNA on hepatic oxidative stress and inflammation in a chemically induced rabbit model of variegate porphyria (A and B) Fold change expression levels of (A) Hepcidin and (B) Hsp70 genes determined by qRT-PCR in the rabbits’ livers are shown. (C and D) Protective efficacy of hPBGD mRNA against lipid peroxidation was determined by (C) urine TBARS excretion over time and (D) quantification of the total peak area of urinary TBARS levels after treatment with hemin and hPBGD-mRNA (days from 17 to 20). Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 against the non-injected group.

Figure 4

Therapeutic efficacy of a single i.v. administration of hPBGD mRNA in a chemically induced rabbit model of variegate porphyria (A and B) Systolic blood pressure at (A) baseline (empty boxes) and during the D8 AIA/rifampicin challenge (full boxes) and (B) after treatment during the D15 challenge. Animals treated with hemin or hPBGD-mRNA regained normal systolic blood pressure. (C) Amplitude of sciatic nerve in an electrophysiological nerve conduction velocity study performed at the end of challenges D8 (without treatment) and D15 (which included treatment). (D) A GTT was performed in order to study insulin resistance in fasted rabbits. Serum glucose levels were measured at times 0, 5, 10, 20, 30, 45, 60, 75, and 90 min post-injection of glucose solution (0.5 g/kg, i.v.). (E) Quantification of the total peak area of the serum glucose levels. (F) Brain ¹⁸F-FDG uptake in 16-h fasted rabbits performed at the end of challenges D8 (without treatment) and D15 (which included treatment). Data are means ± SD. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 versus the non-injected group.