Systemic messenger RNA replacement therapy is effective in a novel clinically relevant model of acute intermittent porphyria developed in non-human primates

Abstract

Objective: Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by haploinsufficiency of hepatic porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthesis. Individuals with AIP experience neurovisceral attacks closely associated with hepatic overproduction of potentially neurotoxic heme precursors.

Design: We replicated AIP in non-human primates (NHPs) through selective knockdown of the hepatic PBGD gene and evaluated the safety and therapeutic efficacy of human PBGD (hPBGD) mRNA rescue.

Results: Intrahepatic administration of a recombinant adeno-associated viral vector containing short hairpin RNA against endogenous PBGD mRNA resulted in sustained PBGD activity inhibition in liver tissue for up to 7 months postinjection. The administration of porphyrinogenic drugs to NHPs induced hepatic heme synthesis, elevated urinary porphyrin precursors and reproduced acute attack symptoms in patients with AIP, including pain, motor disturbances and increased brain GABAergic activity. The model also recapitulated functional anomalies associated with AIP, such as reduced brain perfusion and cerebral glucose uptake, disturbances in hepatic TCA cycle, one-carbon metabolism, drug biotransformation, lipidomic profile and abnormal mitochondrial respiratory chain activity. Additionally, repeated systemic administrations of hPBGD mRNA in this AIP NHP model restored hepatic PBGD levels and activity, providing successful protection against acute attacks, metabolic changes in the liver and CNS disturbances. This approach demonstrated better efficacy than the current standards of care for AIP.

Conclusion: This novel model significantly expands our understanding of AIP at the molecular, biochemical and clinical levels and confirms the safety and translatability of multiple systemic administration of hPBGD mRNA as a potential aetiological AIP treatment.

Keywords: DRUG METABOLISM; ENERGY METABOLISM; GENE THERAPY; HEPATOBILIARY DISEASE.

Figure 1. Schematic timeline representation of the study and biochemical status and gene expression of heme synthesis pathway. (A) Schematic timeline representation of the characterisation of the AIP model in NHPs. The rAAV8 vector was administered directly via the hepatic blood flow at a dosage of 4.18×10¹³ gc/kg BW. To replicate acute attacks, NHPs were administered with porphyrinogenic drugs, starting 4 weeks after the rAAV injection. Given that acute barbiturate challenge can transiently alter brain perfusion, affect the insulin signalling pathway in the brain, and enhance inhibitory GABAergic neurotransmission, a separate protocol was employed before positron-emission tomography (PET) and cerebral blood flow (CBF) studies. One male AIP NHP received a total of 5 doses of givosiran (2.5 mg/kg, administered s.c. every three 3 weeks; Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA). Another male AIP NHP was treated with 19 doses of hemin (2 mg/kg intravenous Normosang, Orphan Europe, Recordati, Milan, Italy), over 6 cycles, with three doseases in each. The cycles were administered every 2-weeks, and an additional dose was given 1 week before sacrifice. Three AIP NHPs (one male) received a total of 7–8 doses of mRNA, with one dose of 0.5 mg/kg of hPBGD mRNA administered every 2 weeks. Lastly, three other AIP NHPs did not receive any treatment. A comprehensive description of the methods used is provided in online supplemental materials and methods. Mean peak urinary excretion of (B) ALA and (C) PBG after repeated acute attacks and multidose administration of current and emerging therapies. (D) Hepatic expression of ALAS1 in NHPs with AIP after treatment (three measurements per animal). (E) ALAS1 immunoblot in liver samples from WT NHPs and NHPs with AIP after recurrent acute attacks and repeated administrations of treatments. (F) Hepatic PBGD activity of NHPs with AIP after treatment (determined in three different lobes per animal). (G) Hepatic expression of HO1 in NHPs with AIP after treatment (three measurements per animal). Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. Data are expressed as mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs WT, untreated or indicated groups. AIP, acute intermittent porphyria; ANOVA, analysis of variance; rAAV, recombinant adeno-associated viral; NHPs, non-human primates; PBGD, porphobilinogen deaminase.

Figure 2. Therapeutic efficacy of multidose intravenous administration of hPBGD mRNA against CNS alterations in AIP NHPs. (A) Representative PET/CT images of [¹¹C]flumazenil brain distribution from an untreated NHP (left) with AIP and an NHP injected with hPBGD mRNA (right) before (baseline) and after (porphyria) PBGD inhibition, and at the end of the study after recurrent acute attack and treatment. (B) Quantification of [¹¹C]flumazenil brain uptake in NHPs at different time points described in A. (C) Representative PET/CT images of [¹⁸F]FDG brain uptake and (D) quantification from untreated NHPs with AIP and animals treated with hPBGD mRNA, hemin and givosiran. (E) Quantification of CBF and (F) representative MRI CBF maps overlaid over the anatomical images of NHPs at baseline, with induced porphyria and after treatment with hPBGD mRNA, hemin and givosiran. Significant heterogeneity was observed among animals in baseline CBF values; however, no gender difference was noted in the detection of hypoperfusion in the model. Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. Data are expressed as mean±SD. *p<0.05; **p<0.01 vs WT, untreated or indicated groups. AIP, acute intermittent porphyria; ANOVA, analysis of variance; CBF, cerebral blood flow; CNS, central nervous system; hPBGD, human porphobilinogen deaminase; NHPs, non-human primates; PET, positron emission tomography.

Figure 3. Therapeutic efficacy of multidose intravenous administration of hPBGD mRNA against peripheral nervous system in AIP NHPs. (A) Amplitude of median, ulnar and plantar sensitive nerve, and (B) velocity of nerve conduction in electrophysiological studies performed after repeated acute attacks and the administration of repeated doses of hPBGD mRNA, hemin and givosiran. Comparisons between two groups were analysed by paired t-tests. Data are expressed as the percentage respect to baseline values (mean±SD). *p<0.05; **p<0.01 vs baseline data. AIP, acute intermittent porphyria; hPBGD, human porphobilinogen deaminase; NHPs, non-human primates.

Figure 4. Transcriptomic analysis of liver-specific genes and therapeutic efficacy of multidose intravenous administration of hPBGD mRNA against metabolomic alterations on the TCA cycle. (A) HeatMap illustrating the expression levels of key hepatic genes critical for metabolic homoeostasis. Transcriptomic analyses were conducted on liver samples of 15 age-matched WT NHPs (including 7 males), and from 3 lobes of three AIP NHPs. Additionally, three liver lobes from each of the three AIP NHPs receiving MD of hPBGD mRNA, three liver lobes from one male NHP who underwent MD hemin administration and another male injected with givosiran were also included. RNA-Seq Data Analysis was performed with a Benjamini-Hochberg correction to get log-adjusted p values, without the loss of precision from undoing and redoing the log-transformations. (B–G) Metabolomic analysis reveals notable differences in TCA metabolites between AIP NHP livers and the impact of treatments. Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. Data correspond to three hepatic lobes from each AIP NHP and are expressed as mean±SD *p<0.05; **p<0.01; ***p<0.001 vs WT animals or indicated groups. AIP, acute intermittent porphyria; ANOVA, analysis of variance; hPBGD, human porphobilinogen deaminase; MD, multidose; NHPs, non-human primates.

Figure 5. Metabolomic study focusing on the OCM and TCA cycle. (A) HeatMap illustrating levels of key hepatic metabolites for OCM and TCA cycles. Analyses were conducted on liver samples of 7 age-matched WT NHPs (including 4 males) and from 3 lobes of three AIP NHPs. Additionally, three liver lobes from each of the three AIP NHPs receiving MD of hPBGD mRNA, three liver lobes from one male NHP who underwent MD hemin administration and another male injected with givosiran were also included. (B-J) Metabolomic analysis reveals notable differences in OCM metabolites between AIP NHP livers and the impact of treatments. Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. Data correspond to three hepatic lobes from each AIP NHP and are expressed as mean±SD *p<0.05; **p<0.01; ***p<0.001 **** p<0.0001 vs WT animals or indicated groups. AIP, acute intermittent porphyria; GPC, glycerophosphocholine; hPBGD, human porphobilinogen deaminase; MD, multidose; NHPs, non-human primates; OCM, one-carbon metabolism; SAMe, S-adenosylmethionine; SAH, S-adenosylhomocysteine; dcSAMe, decarboxylated S-adenosylmethionine; MTA, 5′-deoxy-5′-methylthioadenosine.

Figure 6. Serum lipidomic analysis of AIP NHPs and therapeutic efficacy of hPBGD mRNA. Serum levels of (A) phosphatidylethanolamines (PE) (36:4) and (B) PE (P-16:0/18:2); (C) Phosphatidylcholines (PC) (38:2) and (D) PC (O-18:0/18:2); (E) Triglycerides (TG) (51:0) and (F) TG(53:1); (G) Cholesterol esters (ChoE)(17:1) and (H) Sphingolipids (SM (43:1)) and (I) SM (d18:1/25:0). Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. Data correspond to different serum samples taken over two last months of the study from each AIP NHP and are expressed as mean±SD. *p<0.05; **p<0.01; ***p<0.001 vs WT animals or indicated groups. AIP, acute intermittent porphyria; ANOVA, analysis of variance; hPBGD, human porphobilinogen deaminase; NHPs, non-human primates.

Figure 7. Therapeutic efficacy of multidose intravenous administration of hPBGD mRNA against some haemoproteins function. We investigated potential alterations in drug metabolism following intragastric administration of a bolus of caffeine and acetaminophen. (A) Pharmacokinetic and (B) area under the curve quantification of caffeine (left) and acetaminophen (right) in the serum of untreated and treated animals. Data correspond to different serum samples taken over two last months of the study from each AIP NHP and are expressed as mean±SD. (C) Cumulative activity of the mitochondrial respiratory complexes I–IV. Given that Complex II-driven respiration is activated by succinyl-CoA, enzymatic activities were measured at the maximum rate with saturated substrates, ensuring independence from the contribution of the TCA cycle. (D) Immunodetection of mitochondrial complex as showed by Western-Blot from liver samples. Data correspond to three hepatic lobes from each AIP NHP and are expressed as mean±SD. Data were analysed using one-way ANOVA. Pairwise comparisons were made using Bonferroni’s multiple comparison tests. *p<0.05; **p<0.01; vs WT animals or indicated groups. AIP, acute intermittent porphyria; ANOVA, analysis of variance; hPBGD, human porphobilinogen deaminase; NHP, non-human primate.