In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1.
We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-1). Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.
Yu
M
M
Department of Biology, University of California at San Diego, La Jolla 92093-0665.
Poeschla
E
E
Yamada
O
O
Degrandis
P
P
Leavitt
M C
MC
Heusch
M
M
Yees
J K
JK
Wong-Staal
F
F
Hampel
A
A
D001483:Base Sequence; D002460:Cell Line; D015684:Gene Products, pol / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000378:metabolism* / Q000494:pharmacology; D012367:RNA, Viral / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology; D014779:Virus Replication / Q000187:drug effects
D015684:Gene Products, pol; D016337:RNA, Catalytic; D012367:RNA, Viral