Daniel Schümperli


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Schümperli

First Name

Daniel

ORCID

0000-0002-9644-134X

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2019 - 201912020 - 20222
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Publications 1 - 3 of 3
  • Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals
    Item type: Book Chapter
    Halloy, François; Brönnimann, Paulina; Hall, Jonathan; et al. (2022)
    Methods in Molecular Biology ~ Alternative Splicing
    We describe methods to follow the fate of oligonucleotides after their injection into experimental animals. The quantitation in various tissues, blood or bone marrow cells is possible by chemical ligation PCR. This method works independently of chemical modifications of the oligonucleotide and/or its conjugations to lipid or peptide moieties. Moreover, metabolization intermediates can be detected by mass spectrometry. Together with a readout assay for the biochemical or physiological effects, which will differ, depending on the particular purpose of the oligonucleotide, these methods allow for a comprehensive understanding of oligonucleotide behavior in a living organism.
  • Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria
    Item type: Journal Article
    Halloy, François; Iyer, Pavithra S.; Ćwiek, Paulina; et al. (2020)
    Nucleic Acids Research
    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.
  • Delta-aminolevulinic acid synthase 2 expression in combination with iron as modifiers of disease severity in erythropoietic protoporphyria
    Item type: Journal Article
    Barman-Aksözen, Jasmin; Halloy, François; Iyer, Pavithra S.; et al. (2019)
    Molecular Genetics and Metabolism
    Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5′UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP.
Publications 1 - 3 of 3