1. Effects of Self-Targeting and Cargo Orientation on Efficient Delivery of Anti-HIV-1 RT RNA Aptamer Cargo By a Lentiviral Vector
Margaret J. Lange1,2, Carolina Camargo1,3, and Donald H. Burke1,2,4
Life Sciences Center1, Department of Molecular Microbiology & Immunology2, Genetics Area Program3, Department of Biochemistry4, University of Missouri, Columbia, Missouri, USA
Aptamers were originally selected against HIV Reverse Transcriptase using SELEX technology (in vitro evolution).
To test many aptamers in a gene therapy context, we modified the pHIV7-GFP lentiviral vector into a Destination vector for use as a Gateway Cloning vector.
Vector transduction was also successful in CEM-T4 cells, with evident self-targeting demonstrating aptamer functionality in terms of transduction efficiency (a) and viral titer (b). 9%
(a) Transduction Efficiency
(b) Viral Titer
HIV RT
Self-targeting can be reduced by addition of excess Gag/Pol, as show below in a Gag/Pol plasmid titration with constant amounts of control or aptamer-expressing vector and VSV-G.
Aptamers obtained from this selection were grouped into families based on their proposed secondary structure.
The pHIV7-GFP Destination vectors were engineered to allow cloning of both forward and reverse orientation aptamer cassettes with a PolyA signal or lacking a PolyA signal.
Aptamers effectively inhibited lentiviral vector transduction in the forward orientation as compared to the arbitrary RNA control. Transfection efficiencies for the vectors were similar.
PolyA Signal
All of the aptamers obtained from the selection were able to inhibit the function of HIV RT in biochemical assays by competing with the viral genome for binding.
No PolyA Signal
However, overall transduction efficiency decreases with increasing Gag/Pol transfection.
Aptamers were tested against a phylogenetically diverse panel of HIV RTs. Some aptamers were found to inhibit many subtypes of RT, while other aptamers demonstrated specificity.
However, reverse orientation vectors demonstrated very low transduction efficiency, suggesting a promoter silencing effect. Notably, transfection efficiencies for the reverse orientation vectors were similar to those in forward orientation.
Importantly, the aptamers inhibited HIV replication in single cycle (a) and multiple cycle (b) infectivity assays. a
Notably, use of the Tet Operator system (CMV-TO) abrogates self-targeting without reduction in overall transduction efficiency.
PolyA Signal
No PolyA Signal
Represssed
Represssed
Producer Cell
10% 9%
12% b
Stable, aptamer-expressing TZM-bl cell lines
Arbitrary RNA Control
F1pk Aptamer
CONCLUSIONS:
Aptamers successfully inhibit HIV replication when expressed in a lentiviral vector context.
Cassette expression in the reverse orientation significantly reduced transduction efficiency, which may be due to a promoter silencing effect,
Self-targeting can be reduced by out-competing aptamer with excess Gag/Pol, but providing excess Gag/Pol reduces transduction efficiency.
Self-targeting can also be effectively reduced by utilizing a regulated promoter system, CMV-TO.
Funding Sources
NIH R01-AI074389
NIH F32-AI085627
@DHBurkeAguero
@BurkeLabRNA
F2pk Aptamer