Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein-polylysine carriers.
We have previously shown targeting of DNA to hepatocytes using an asialoorosomucoid-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-ligand interactions. In these studies, our objective was to determine if AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmid or single-stranded oligonucleotide DNA, alone or bound to conjugate, was incubated under conditions mimicking those encountered during in vitro and in vivo transfections. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded oligonucleotides was inhibited 3- to 6-fold in serum during 5 hours of incubation. For complexed plasmids, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmid was completely degraded after 15 minutes in serum or 60 minutes in medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercoiled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation.
Chiou
H C
HC
Gene Therapy Group, TargeTech Inc., Carlsbad, CA 92008.
Tangco
M V
MV
Levine
S M
SM
Robertson
D
D
Kormis
K
K
Wu
C H
CH
Wu
G Y
GY
D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry*; D001769:Blood; D002417:Cattle; D002457:Cell Extracts; D003470:Culture Media; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004337:Drug Carriers / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011107:Polylysine / Q000031:analogs & derivatives* / Q000737:chemistry; D051381:Rats
D001212:Asialoglycoproteins; D002457:Cell Extracts; D003470:Culture Media; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004337:Drug Carriers; D009961:Orosomucoid; D011089:Polydeoxyribonucleotides; C086457:asialoglycoprotein-polylysine conjugate; C017030:asialoorosomucoid; D011107:Polylysine; D003851:Deoxyribonucleases