title: Low RNA translation activit limits the efficacy of hydrodynamic gene transfer to pig liver in vivo.
abstract: BACKGROUND
  Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo.
  
  
  METHODS
  A solution of enhanced green fluorescent protein (eGFP) DNA was injected under two different venous injection conditions (anterograde and retrograde), employing flow rates of 10 and 20 ml/s in each case, with the aim of identifying the best gene transfer conditions. The gene delivery and information decoding steps were evaluated by measuring the eGFP DNA, mRNA and protein copy number 24 h after transfection. In addition, gold nanoparticles (diameters of 4 and 15 nm) were retrogradely injected (10 ml/s) to observe, by electron microscopy, the ability of the particle to access the hepatocyte.
  
  
  RESULTS
  The gene delivery level was higher with anterograde injection, whereas the efficacy of gene expression was better with retrograde injection, suggesting differences in the decoding processes. Thus, retrograde injection mediates gene transcription (mRNA copy/cell) equivalent to that of intermediate expression proteins but the mRNA translation was lower than that of rare proteins. Electron microscopy showed that nanoparticles within the hepatocyte were almost exclusively 4 nm in diameter.
  
  
  CONCLUSIONS
  The results suggest that the low activity of mRNA translation limits the final efficacy of the gene transfer procedure. On the other hand, the gold nanoparticles study suggests that elongated DNA conformation could offer advantages in that the access of 15-nm particles is very limited.
authors.affiliation: Departamento de Farmacologia, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Sendra
authors.forename: Omar
authors.initials: O
authors.lastname: Carreño
authors.forename: Antonio
authors.initials: A
authors.lastname: Miguel
authors.forename: Eva
authors.initials: E
authors.lastname: Montalvá
authors.forename: María José
authors.initials: MJ
authors.lastname: Herrero
authors.forename: Francisco
authors.initials: F
authors.lastname: Orbis
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Noguera
authors.forename: Domingo
authors.initials: D
authors.lastname: Barettino
authors.forename: Rafael
authors.initials: R
authors.lastname: López-Andújar
authors.forename: Salvador F
authors.initials: SF
authors.lastname: Aliño
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D005260:Female; D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism / Q000648:ultrastructure; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D053768:Metal Nanoparticles / Q000737:chemistry; D010316:Particle Size; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D034421:Sus scrofa; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*
chemical_list: D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006046:Gold
keywords: gene therapy; hydrodynamic; liver; nanoparticle; nonviral; pig