TY  - JOUR
A1  - Lehto, Taavi
A1  - Alvarez, Alejandra Castillo
A1  - Gauck, Sarah
A1  - Gait, Michael J.
A1  - Coursindel, Thibault
A1  - Wood, Matthew J. A.
A1  - Lebleu, Bernard
A1  - Boisguerin, Prisca
T1  - Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells
JF  - Nucleic acids research
N2  - Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy-and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the predominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.
Y1  - 2014
U6  - https://doi.org/10.1093/nar/gkt1220
SN  - 0305-1048
SN  - 1362-4962
VL  - 42
IS  - 5
SP  - 3207
EP  - 3217
PB  - Oxford Univ. Press
CY  - Oxford
ER  - 
TY  - JOUR
A1  - Daskalow, Katjana
A1  - Boisguerin, Prisca
A1  - Jandrig, Burkhard
A1  - van Landeghem, Frank K. H.
A1  - Volkmer, Rudolf
A1  - Micheel, Burkhard
A1  - Schenk, Jörg A.
T1  - Generation of an antibody against the protein phosphatase 1 inhibitor KEPI and characterization of the epitope
N2  - A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 mu M. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues.
Y1  - 2010
UR  - http://ar.iiarjournals.org/
SN  - 0250-7005
ER  -