Selection of potent non-toxic inhibitory sequences from a randomized HIV-1 specific lentiviral short hairpin RNA library.
RNA interference (RNAi) has been considered as an efficient therapeutic approach against the human immunodeficiency virus type 1 (HIV-1). However, to establish a durable inhibition of HIV-1, multiple effective short hairpin RNAs (shRNAs) need to be stably expressed to prevent the emergence of viral escape variants. In this study, we engineered a randomized lentiviral H1-promoter driven shRNA-library against the viral genome. Potent HIV-1 specific shRNAs were selected by ganciclovir treatment of cell lines stably expressing the cDNA of Herpes Simplex Virus thymidine kinase (HSV-TK) fused to HIV-1 nucleotide sequences. More than 50% of 200 selected shRNAs inhibited an HIV-1 based luciferase reporter assay by more than 70%. Stable expression of some of those shRNAs in an HIV-1 permissive HeLa cell line inhibited infection of wild-type HIV-1 by more than 90%. The combination of a randomized shRNA-library directed against HIV-1 with a live cell selection procedure yielded non-toxic and highly efficient HIV-1 specific inhibitory sequences that could serve as valuable candidates for gene therapy studies.
Institute for Medical Microbiology, Immunology and Hygiene, University Hospital Cologne, Cologne, Germany.
Carola
C
Pongratz
Benjamin
B
Yazdanpanah
Hamid
H
Kashkar
Maik J
MJ
Lehmann
Hans-Georg
HG
Kräusslich
Martin
M
Krönke
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D018076:DNA, Complementary; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012367:RNA, Viral / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral