Systemic messenger RNA as an etiological treatment for acute intermittent porphyria

Abstract

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Kinetics of PBGD expression and therapeutic efficacy of a single i.v. injection of hPBGD mRNA administered at the peak of a phenobarbital-induced acute attack in AIP mice.
Fig. 2: Therapeutic efficacy of a single i.v. administration of hPBGD mRNA to prevent a phenobarbital-induced acute attack in AIP mice.
Fig. 3: Therapeutic efficacy of a single i.v. administration of 0.5 mg kg−1 hPBGD mRNA against one or three consecutive phenobarbital-induced acute attacks in AIP mice.
Fig. 4: Therapeutic efficacy of multi-dose i.v. administration of hPBGD mRNA against three consecutive phenobarbital-induced acute attacks in AIP mice.
Fig. 5: Therapeutic efficacy of a single dose or multiple doses of hPBGD mRNA against AIA- and rifampicin-induced accumulation of porphyrin precursors in rabbits.
Fig. 6: Hepatic PBGD activity 24 h after single or repeated administration of hPBGD mRNA (0.5 mg kg−1) in female NHPs.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

References

  1. 1.

    Anderson, K. E. et al. Recommendations for the diagnosis and treatment of the acute porphyrias. Ann. Intern. Med. 142, 439–450 (2005).

    Article  PubMed  Google Scholar 

  2. 2.

    Bissell, D. M., Anderson, K. E. & Bonkovsky, H. L. Porphyria. N. Engl. J. Med. 377, 862–872 (2017).

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Fratz, E. J., Stojanovski, B. M., Ferreira G. C. in Handbook of Porphyrin Science Vol. 26 (eds Kadish, K. M. et al.) 3–78 (World Scientific Publishing, Hackensack, NJ, USA, 2014).

  4. 4.

    Harper, P. & Sardh, E. Management of acute intermittent porphyria. Expert Opin. Orphan Drugs 2, 349–368 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Puy, H., Gouya, L. & Deybach, J. C. Porphyrias. Lancet 375, 924–937 (2010).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Marsden, J. T. et al. Audit of the use of regular haem arginate infusions in patients with acute porphyria to prevent recurrent symptoms. JIMD Rep. 22, 57–65 (2015).

    Article  PubMed Central  PubMed  Google Scholar 

  7. 7.

    Handschin, C. et al. Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1α. Cell 122, 505–515 (2005).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Bonkovsky, H. L. et al. Acute porphyrias in the USA: features of 108 subjects from porphyrias consortium. Am. J. Med. 127, 1233–1241 (2014).

    Article  PubMed Central  PubMed  Google Scholar 

  9. 9.

    Bissell, D. M., Lai, J. C., Meister, R. K. & Blanc, P. D. Role of delta-aminolevulinic acid in the symptoms of acute porphyria. Am. J. Med. 128, 313–317 (2015).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Herrick, A. L., Moore, M. R., McColl, K. E. L., Cook, A. & Goldberg, A. Controlled trial of haem arginate in acute hepatic porphyria. Lancet 333, 1295–1297 (1989).

    Article  Google Scholar 

  11. 11.

    Meyer, U. A., Schuurmans, M. M. & Lindberg, R. L. Acute porphyrias: pathogenesis of neurological manifestations. Semin. Liver. Dis. 18, 43–52 (1998).

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Pallet, N. et al. High prevalence of and potential mechanisms for chronic kidney disease in patients with acute intermittent porphyria. Kidney Int. 88, 386–395 (2015).

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Bylesjö, I., Wikberg, A. & Andersson, C. Clinical aspects of acute intermittent porphyria in northern Sweden: a population-based study. Scand. J. Clin. Lab. Invest. 69, 612–618 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Tchernitchko, D. et al. A variant of peptide transporter 2 predicts the severity of porphyria-associated kidney disease. J. Am. Soc. Nephrol. 28, 1924–1932 (2017).

    Article  PubMed  Google Scholar 

  15. 15.

    Willandt, B. et al. Liver fibrosis associated with iron accumulation due to long-term heme-arginate treatment in acute intermittent porphyria: a case series. JIMD Rep 25, 77–81 (2016).

    Article  PubMed  Google Scholar 

  16. 16.

    Schmitt, C. et al. Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver. J. Intern. Med. 284, 78–91 (2018).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Yasuda, M. et al. RNAi-mediated silencing of hepatic Alas1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice. Proc. Natl Acad. Sci. USA 111, 7777–7782 (2014).

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Alnylam reports positive initial clinical results for ALN-AS1, an investigational RNAi therapeutic targeting aminolevulinic acid synthase 1 (ALAS1) for the treatment of acute hepatic porphyrias. Alnylam Pharmaceuticals http://www.businesswire.com/news/home/20150915005532/en/ (2015).

  19. 19.

    Unzu, C. et al. Sustained enzymatic correction by rAAV-mediated liver gene therapy protects against induced motor neuropathy in acute porphyria mice. Mol. Ther. 19, 243–250 (2011).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  20. 20.

    Yasuda, M. et al. AAV8-mediated gene therapy prevents induced biochemical attacks of acute intermittent porphyria and improves neuromotor function. Mol. Ther. 18, 17–22 (2010).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Unzu, C. et al. Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression. Gene Ther. 22, 856–865 (2015).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    D’Avola, D. et al. Phase I open label liver-directed gene therapy clinical trial for acute intermittent porphyria. J. Hepatol. 65, 776–783 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Sahay, G. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31, 653–658 (2013).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  24. 24.

    Sabnis, S. et al. A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 26, 1509–1519 (2018).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    An, D. et al. Systemic messenger RNA therapy as a treatment for methylmalonic acidemia. Cell Rep. 21, 3548–3558 (2017).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Lindberg, R. L. et al. Porphobilinogen deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria. Nat. Genet. 12, 195–199 (1996).

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Gouya, L. et al. EXPLORE: A prospective, multinational, natural history study of patients with acute hepatic porphyria (AHP) with recurrent attacks. ICPP http://www.alnylam.com/wp-content/uploads/2017/06/ICPP-2017-EXPLORE-Presentation-Capella.pdf (2017).

  28. 28.

    Langford, D. J. et al. Coding of facial expressions of pain in the laboratory mouse. Nat. Methods 7, 447–449 (2010).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Matsumiya, L. C. et al. Using the Mouse Grimace Scale to reevaluate the efficacy of postoperative analgesics in laboratory mice. J. Am. Assoc. Lab. Anim. Sci. 51, 42–49 (2012).

    CAS  PubMed Central  PubMed  Google Scholar 

  30. 30.

    Vijayasarathy, C., Damle, S., Lenka, N. & Avadhani, N. G. Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex. Eur. J. Biochem. 266, 191–200 (1999).

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Atamna, H., Liu, J. & Ames, B. N. Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts: revelance to aging. J. Biol. Chem. 276, 48410–48416 (2001).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Atamna, H., Killilea, D. W., Killilea, A. N. & Ames, B. N. Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging. Proc. Natl Acad. Sci. USA 99, 14807–14812 (2002).

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Homedan, C. et al. Acute intermittent porphyria causes hepatic mitochondrial energetic failure in a mouse model. Int. J. Biochem. Cell Biol. 51, 93–101 (2014).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Bonkowsky, H. L., Tschudy, D. P., Weinbach, E. C., Ebert, P. S. & Doherty, J. M. Porphyrin synthesis and mitochondrial respiration in acute intermittent porphyria: studies using cultured human fibroblasts. J. Lab. Clin. Med. 85, 93–102 (1975).

    CAS  PubMed  Google Scholar 

  35. 35.

    Xie, L. et al. Age- and sex-based hematological and biochemical parameters for Macaca fascicularis. PLoS ONE 8, e64892 (2013).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  36. 36.

    Kim, C. Y. et al. Hematological and serum biochemical values in cynomolgus monkeys anesthetized with ketamine hydrochloride. J. Med. Primatol. 34, 96–100 (2005).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Sedic, M. et al. Safety evaluation of lipid nanoparticle-formulated modified mRNA in the Sprague–Dawley rat and cynomolgus monkey. Vet. Pathol. 55, 341–354 (2018).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Dowman, J. K. et al. Liver transplantation for acute intermittent porphyria is complicated by a high rate of hepatic artery thrombosis. Liver Transpl. 18, 195–200 (2012).

    Article  PubMed Central  PubMed  Google Scholar 

  39. 39.

    Rogers, G. W. et al. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS ONE 6, e21746 (2011).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  40. 40.

    Anderson, P. M. & Desnick, R. J. Porphobilinogen deaminase: methods and principles of the enzymatic assay. Enzyme 28, 146–157 (1982).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Goldberg, A. & Rimington, C. Experimentally produced porphyria in animals. Proc. R. Soc. Lond. B. 143, 257–279 (1955).

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Klinger, W. & Muller, D. The influence of allyl isopropyl acetamide on d-aminolevulinic acid synthetase and cytochrome P-450. Acta Biol. Med. Ger. 39, 107–112 (1980).

    CAS  PubMed  Google Scholar 

  43. 43.

    Tokola, O., Linden, I. B. & Tenhunen, R. The effects of haem arginate and haematin upon the allylisopropylacetamide induced experimental porphyria in rats. Pharmacol. Toxicol. 61, 75–78 (1987).

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Muller-Eberhard, U., Eiseman, J. L., Foidart, M. & Alvares, A. P. Effect of heme on allylisopropylacetamide-induced changes in heme and drug metabolism in the rhesus monkey (Macaca mulatta). Biochem. Pharmacol. 32, 3765–3769 (1983).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    McColl, K. E. et al. Effect of rifampicin on haem and bilirubin metabolism in man. Br. J. Clin. Pharmacol. 23, 553–559 (1987).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  46. 46.

    Innala, E., Backstrom, T., Bixo, M. & Andersson, C. Evaluation of gonadotropin-releasing hormone agonist treatment for prevention of menstrual-related attacks in acute porphyria. Acta Obstet. Gynecol. Scand. 89, 95–100 (2010).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

P.B., M.A.A. and A.Fo. thank J. Prieto for his enthusiastic and continuous support of our research on porphyria. We thank S. Arcelus and I. Alkain for technical assistance; J. L. Lanciego, A. Rico, L. Guembe and A. Benito for their helpful technical and scientific support with NHPs; P. Harper and E. Sardh for supplying liver explants from patients with porphyria. T1 and T2 mouse strains were provided by U. A. Meyer (Biozentrum of University of Basel, Basel, Switzerland). This study was supported in part by grants from the Spanish Fundación Mutua Madrileña, Spanish Fundación Eugenio Rodríguez Pascual and Spanish Institute of Health Carlos III (FIS) cofinanced by European FEDER funds (grant numbers PI09/02639, PI12/02785, PI15/01951 and PI16/00668 funds). P.B. is supported by a Miguel Servet II (CPII15/00004) contract from Instituto de Salud Carlos III.

Author information

Affiliations

Authors

Contributions

L.J., P.B., L.T.G., C.M.L., M.A.A., P.G.V.M. and A.Fo. designed in vitro and animal experiments. L.J., P.B., D.J., A.S., A.Fr., J.-S.P., X.Z. and A.Fo. performed the experiments and processed animal samples and tissues. D.J., A.S., and A.Fo. performed behavior assays in AIP mice and rabbits. E.S. and A.S. performed mitochondrial function studies. K.B. performed IHC analysis. M.A. and A.Fo. performed electrophysiological studies in AIP mice and rabbits. A.P., A.S., D.J. and A.Fo. performed blood pressure studies. L.J., L.T.G., K.E.B., M.Ka., W.B., S.S., E.S.K., T.S., M.Ke., C.M.L. and P.G.V.M. designed and produced mRNA formulations. L.J., L.T.G., C.M.L., A.Fo. and P.G.V.M. supervised mRNA production and supported administrative, technical and logistic tasks for sending and receiving samples. L.J., P.B., L.T.G. and A.Fo. performed all statistical analyses. L.J., P.B., L.T.G., A.S., D.J., E.S., M.A., A.P., P.G.V.M. and A.Fo. analyzed the data. L.J., P.B., L.T.G., M.A.A., C.M.L., P.G.V.M. and A.Fo. wrote the manuscript, assisted by A.S. and D.J for figures and tables. All authors performed a critical revision of the manuscript for important intellectual content and final approval of the manuscript.

Corresponding authors

Correspondence to Paolo G. V. Martini or Antonio Fontanellas.

Ethics declarations

Competing interests

L.J., L.T.G., A.Fr., K.E.B., K.B., M.Ka., W.B., J.-S.P., X.Z., S.S., E.S.K., T.S., M.Ke., C.M.L. and P.G.V.M. are employees of Moderna Therapeutics, Inc. focusing on the development of therapeutic approaches for rare diseases. The other authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1–5 and Supplementary Methods

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jiang, L., Berraondo, P., Jericó, D. et al. Systemic messenger RNA as an etiological treatment for acute intermittent porphyria. Nat Med 24, 1899–1909 (2018). https://doi.org/10.1038/s41591-018-0199-z

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing