title: Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters.
abstract: Small interfering RNA (siRNA) mediates sequence-specific RNA cleavage and represents a potential approach to treat the infection of human immunodeficiency virus (HIV). Expression of a single siRNA species frequently led to the emergence of HIV escape variants. Thus, multiple siRNAs targeted to different regions in the HIV-1 genome may be required. However, overexpression of different anti-HIV siRNA genes from multiple pol III promoters can induce cell toxicity, thus may not be a viable option in the setting of human gene therapy trials. In the current study, we evaluated the strategy of using pol II promoters to drive the expression of siRNAs against HIV-1. We replaced the stem sequence in the stem-loop structure of the well-characterized miR-30a with siRNA sequences and showed that designed microRNA (miRNA) could be expressed from pol II promoters. We demonstrated efficient inhibition of HIV-1 replication with such designed miRNA, but the efficacy was directly correlated with the expression level. Both the vector copy number and the promoter strength directly affected the ability of the siRNA to inhibit HIV-1 replication. We also showed that a combination of pol II and pol III promoters to express two different siRNAs increased the efficacy against HIV-1 replication without comprising cell viability.
authors.affiliation: Division of Virology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: H-L
authors.initials: HL
authors.lastname: Lo
authors.forename: T
authors.initials: T
authors.lastname: Chang
authors.forename: P
authors.initials: P
authors.lastname: Yam
authors.forename: P M
authors.initials: PM
authors.lastname: Marcovecchio
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: J A
authors.initials: JA
authors.lastname: Zaia
authors.forename: J-K
authors.initials: JK
authors.lastname: Yee
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D015658:HIV Infections / Q000628:therapy*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D035683:MicroRNAs / Q000008:administration & dosage*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics*; D012320:RNA Polymerase III / Q000235:genetics; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics