538. Promoter Compatibility Enables Viral Vectors to Deliver Antiviral RNA Genes

Abstract

HIV-1 reverse transcriptase (RT) copies the viral RNA genome into double-stranded DNA during viral replication. Nucleic acid aptamers that bind RT inhibit its enzymatic activity in biochemical and biological assays and can be delivered to target cells by lentiviral vectors. We have demonstrated that RNA aptamers efficiently suppress viral replication; however, using lentiviral vectors to deliver anti-HIV genes might give rise to self-targeting, in which expression of the cargo gene during vector production interferes with efficient gene delivery. We hypothesize that promoter strength will predict the degree of viral suppression, and that regulated promoters are more suitable than constitutive promoters for delivering anti-RT aptamer genes by viral vectors. Specifically, promoters that are strong in T cells are expected to yield the most effective suppression of replicating virus, while promoters that are weak in HEK293FT are expected to yield the highest transduction efficiencies by minimizing self-targeting during particle production.To establish relative promoter strength in producer HEK293FT cells, we first cloned constitutive and regulated promoters (CMV, CMV-TO, EF1α, UbC, CD4 and U6) to drive the expression of a fluorescent aptamer (stabilized dimeric Broccoli) and EGFP. Baseline promoter strength (EGFP MFI) varied widely in HEK293FT cells, with strong expression under CMV, intermediate expression under EF1α and UbC, almost no expression under CD4. Regulated expression was achievable under CMV-TO by co-transfecting a plasmid expressing TetR Repressor.We next evaluated the effect of each promoter/aptamer combination on viral infectivity for which constructs were transiently transfected into producer cells (HEK293FT) along with proviral and helper plasmids. Virus was harvested after 48h and used in subsequent infections. Virus produced in aptamer-expressing HEK293 FT cells showed decrease in viral infectivity both in epithelial (HEK293FT) and T cell lines (CEM-T4), in each case correlating with promoter strength in the producer cells. These data strongly suggest that promoters with lower levels of expression in producer cells, such as CD4 and CMV-TO, might overcome self-targeting and become optimal for production of lentiviral vectors, which is an important goal in order to move aptamer-based strategies into pre-clinical contexts. Additionally, EGFP and stabilized dimeric Broccoli expression are informative surrogates for viral inhibition.