Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Alexandre P A Theocharides,Alice Ghidini,Daniel Schümperli,Elisabeth I Minder,François Halloy,Jasmin Barman-Aksözen,Jonathan Hall,Nicole Wildner-Verhey Van Wijk,Paulina Ćwiek,Pavithra S Iyer,Xiaoye Schneider-Yin

doi:10.1093/nar/gkaa229

Alexandre P A Theocharides, Alice Ghidini + Show 9 more

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https://doi.org/10.1093/nar/gkaa229

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Abstract

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Highlights

Erythropoietic protoporphyria (EPP; OMIM # 177000) is a rare autosomal recessive disorder [1], that is caused by deficiency in the heme biosynthesis enzyme ferrochelatase (FECH, EC 4.99.1.1) (Figure 1A) [2,3]
With emetine treatment to inhibit nonsense-mediated mRNA decay (NMD) of mRNA in lymphoblasts immortalized from donors, we found approximately 30% and 60% of the FECH transcripts were aberrantly spliced for the T/T (c.315-48T)- and C/C (c.315-48C)-homozygotes, respectively
The drug is typically optimized to provide a maximum inhibition of the target; for the former, the objective is to increase expression of the target to counter a loss of function

Summary

Introduction

Erythropoietic protoporphyria (EPP; OMIM # 177000) is a rare autosomal recessive disorder [1], that is caused by deficiency in the heme biosynthesis enzyme ferrochelatase (FECH, EC 4.99.1.1) (Figure 1A) [2,3]. With over 80% of heme produced in red blood cells progenitors, the bone marrow is the major site of biosynthesis. Kidneys and liver are minor sites of heme biosynthesis [4]. Upon short exposure to visible light, PPIX produces reactive oxygen species leading to lipid peroxidation, cell membrane damage and inflammation. This causes patients to suffer from burn-like injuries of the endothelial cells of the blood vessels and the surrounding tissue, associated with acute severe pain; some will develop hepatic complications with deposits of PPIX crystals and are at risk of liver failure [5,6]

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