Ramezani Ali, Ma Xue Zhong, Ameli Masoud, Arora Alka, Joshi Sadhna
Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
Front Biosci. 2006 Sep 1;11:2940-8. doi: 10.2741/2023.
A combination gene therapy strategy using an ASPsi-gag antisense RNA (targeted against the packaging signal and the gag-coding region) and a multimeric hammerhead ribozyme Rz1-9 (targeted against nine sites within the env-coding region) or Rz1-14 (targeted against 14 sites within the 5' leader and the pro-, pol-, vif- and env-coding regions) was assessed for inhibiting HIV-1 replication. A murine stem cell virus (MSCV)-based MGIN vector was used to express Rz1-9, Rz1-14, ASPsi-gag, Rz1-9ASPsi-gag, or Rz1-14ASPsi-gag RNA in a CD4+ T lymphoid cell line. Stable transductants were shown to express similar levels of interfering RNA. HIV-1 replication was inhibited in cells expressing Rz1-9 and Rz1-14. Little inhibition of HIV-1 replication was observed in cells expressing ASPsi-gag RNA. Thus, the multimeric hammerhead ribozymes inhibit HIV-1 replication better than the antisense RNA. Inhibition of HIV-1 replication in cells expressing Rz1-9ASPsi-gag or Rz1-14ASPsi-gag RNA was worse than that obtained with the multimeric ribozymes alone. This result suggests that co-expression of antisense RNA decreases the anti-HIV potential of ribozymes. The multimeric ribozymes and the antisense RNA were designed to target different sites within the HIV-1 RNA. They are not expected to interact with each other. Neither are they expected to compete with each other for binding to the HIV-1 RNA. Instead, the antisense RNA binding to its (1553 nt-long) target site may have resulted in a decreased ribozyme turn over. Furthermore, since the antisense RNA/HIV-1 RNA hybrids are degraded by the cells, the co-expressed antisense RNA may have led to ribozyme degradation.
一种联合基因治疗策略，使用ASPsi - gag反义RNA（靶向包装信号和gag编码区）和多聚锤头状核酶Rz1 - 9（靶向env编码区内的9个位点）或Rz1 - 14（靶向5'前导序列以及pro、pol、vif和env编码区内的14个位点），用于评估其对HIV - 1复制的抑制作用。基于小鼠干细胞病毒（MSCV）的MGIN载体用于在CD4 + T淋巴细胞系中表达Rz1 - 9、Rz1 - 14、ASPsi - gag、Rz1 - 9ASPsi - gag或Rz1 - 14ASPsi - gag RNA。稳定转导子显示表达相似水平的干扰RNA。在表达Rz1 - 9和Rz1 - 14的细胞中，HIV - 1复制受到抑制。在表达ASPsi - gag RNA的细胞中，未观察到对HIV - 1复制的明显抑制。因此，多聚锤头状核酶比反义RNA能更好地抑制HIV - 1复制。在表达Rz1 - 9ASPsi - gag或Rz1 - 14ASPsi - gag RNA的细胞中，对HIV - 1复制的抑制作用比单独使用多聚核酶时更差。这一结果表明反义RNA的共表达降低了核酶的抗HIV潜力。多聚核酶和反义RNA被设计为靶向HIV - 1 RNA内的不同位点。预计它们不会相互作用。也预计它们不会相互竞争与HIV - 1 RNA的结合。相反，反义RNA与其（1553个核苷酸长）靶位点的结合可能导致核酶周转减少。此外，由于反义RNA/HIV - 1 RNA杂交体被细胞降解，共表达的反义RNA可能导致核酶降解。
