Efficient nuclear delivery of antisense oligodeoxynucleotides and selective inhibition of CETP expression by apo E peptide in a human CETP-stably transfected CHO cell line.
N,N-Dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP-stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.
Liu
K
K
Departments of Internal Medicine, Fukuoka University School of Medicine,Fukuoka,Japan.
Ou
J
J
Saku
K
K
Jimi
S
S
Via
D P
DP
Sparrow
J T
JT
Zhang
B
B
Pownall
H J
HJ
Smith
L C
LC
Arakawa
K
K
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics / Q000494:pharmacology*; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053480:Cholesterol Ester Transfer Proteins; D006224:Cricetinae; D003470:Culture Media / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D001057:Apolipoproteins E; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D003470:Culture Media; D006023:Glycoproteins; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D012333:RNA, Messenger