The influence of sodium glycocholate and other additives on the in vivo transfection of plasmid DNA in the lungs.
OBJECTIVE
A plasmid containing the luciferase 'marker' cDNA was constructed to test non viral gene delivery formulations in vivo.


METHODS
A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200-250 microl of solution containing 200 microg plasmid +/- lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay.


RESULTS
The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone > or = lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (approximately 16 pg) using the polymers (approximately 1.5 pg) to approximately 125 times greater than the control using the permeation enhancer (approximately 2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1% w/v.


CONCLUSIONS
The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.
Freeman
D J
DJ
Amgen Inc., Thousand Oaks, California 91320, USA.
Niven
R W
RW
D000818:Animals; D002756:Cholagogues and Choleretics / Q000494:pharmacology*; D002784:Cholesterol / Q000031:analogs & derivatives / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification*; D018014:Gene Transfer Techniques; D006000:Glycocholic Acid / Q000494:pharmacology*; D000882:Haplorhini; D006801:Humans; D007668:Kidney / Q000166:cytology; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000502:physiology*; D008297:Male; D008722:Methods; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*
D002756:Cholagogues and Choleretics; D018076:DNA, Complementary; D008081:Liposomes; D012333:RNA, Messenger; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D002784:Cholesterol; D008156:Luciferases; D006000:Glycocholic Acid