TY - JOUR
T1 - mCherry on Top
T2 - A Positive Read-Out Cellular Platform for Screening DMD Exon Skipping Xenopeptide-PMO Conjugates
AU - Lessl, Anna-Lina
AU - Pöhmerer, Jana
AU - Lin, Yi
AU - Wilk, Ulrich
AU - Höhn, Miriam
AU - Hörterer, Elisa
AU - Wagner, Ernst
AU - Lächelt, Ulrich
N1 - Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.
PY - 2023/12/20
Y1 - 2023/12/20
N2 - Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new in vitro-in vivo screening platform based on mdx exon 23 skipping. Here, a new in vitro positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations in vitro was then translated to in vivo trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned in vitro-in vivo workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO-XP conjugate formulation was found to induce highly potent exon skipping in vitro and redistributed PMO activity in different organs in vivo.
AB - Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new in vitro-in vivo screening platform based on mdx exon 23 skipping. Here, a new in vitro positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations in vitro was then translated to in vivo trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned in vitro-in vivo workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO-XP conjugate formulation was found to induce highly potent exon skipping in vitro and redistributed PMO activity in different organs in vivo.
UR - https://www.scopus.com/pages/publications/85179812115
U2 - 10.1021/acs.bioconjchem.3c00408
DO - 10.1021/acs.bioconjchem.3c00408
M3 - Article
C2 - 37991502
SN - 1043-1802
VL - 34
SP - 2263
EP - 2274
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 12
ER -