Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions.
BACKGROUND
HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system.


METHODS
Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined.


RESULTS
RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed.


CONCLUSIONS
Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.
ter Brake
Olivier
O
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, The Netherlands.
Berkhout
Ben
B
D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D003062:Codon / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics; D006678:HIV / Q000187:drug effects*; D006801:Humans; D016086:Lentivirus / Q000187:drug effects / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012367:RNA, Viral; D014161:Transduction, Genetic
D003062:Codon; D015685:Fusion Proteins, gag-pol; D015694:Gene Products, rev; D034741:RNA, Small Interfering; D012367:RNA, Viral