title: Induction of splice correction by cell-penetrating peptide nucleic acids.
abstract: BACKGROUND
  Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.
  
  
  METHODS
  HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.
  
  
  RESULTS
  All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.
  
  
  CONCLUSIONS
  These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.
authors.affiliation: Department of Neurochemistry, Stockholm University, S. Arrheniusv 21A, SE-10691 Stockholm, Sweden.
authors.forename: Samir
authors.initials: S
authors.lastname: El-Andaloussi
authors.forename: Henrik J
authors.initials: HJ
authors.lastname: Johansson
authors.forename: Pontus
authors.initials: P
authors.lastname: Lundberg
authors.forename: Ulo
authors.initials: U
authors.lastname: Langel
mesh_terms: D002463:Cell Membrane Permeability / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology; D018014:Gene Transfer Techniques; D030342:Genetic Diseases, Inborn / Q000628:therapy; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects; D020135:Peptide Nucleic Acids / Q000009:adverse effects / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014161:Transduction, Genetic / Q000379:methods; D016896:Treatment Outcome
chemical_list: D020135:Peptide Nucleic Acids; D022821:RNA Splice Sites; D002738:Chloroquine