548. Cyclophilin A as a Cellular Target for Gene Therapy of HIV-1

Abstract

Cyclophilin A (CYPA), one of several ubiquitously distributed intracellular proteins that possess peptidyl-proloyl cis-trans isomerase activity, is specifically incorporated into newly formed HIV-1 virions through interactions with the Gag polyprotein. When the Gag-CYPA interaction is disrupted, either by pharmacological or genetic manipulation, HIV-1 replication is inhibited. Moreover, virions produced from genetically modified CYPA-/- cells are less infectious, making CYPA a unique cellular target for HIV-1 therapy. We used RNA interference (RNAi) to knockdown expression of endogenous CYPA and determined the effect on HIV-1 infectivity. As a first step towards designing an effective RNAi strategy, we probed the accessibility of the 753-nt CYPA mRNA using an oligonucleotide scanning technique. One of three 21-mer oligonucleotides, predicted to be accessible using free-energy based folding algorithm, promoted endogenous RNase H-mediated cleavage. A short hairpin RNA (shRNA) was derived from this sequence and cloned into a plasmid under a U6+1 promoter. Upon transient transfection of HEK-293 cells, CYPA gene expression was reduced more than 80% (>80%), as measured by quantitative real-time RT-PCR. Depleting HEK-293 cells of endogenous CYPA did not produce observable toxic effects, consistent with previous work that CYPA is not essential for the viability of human cells. We performed cotransfection experiments with the HIV-1 proviral clone pNL4-3, and measured both infectious virion output and infectivity in the presence or absence of CYPA shRNA expression. Virions produced in the presence of CYPA shRNA were equally infectious as virions produced in the absence of CYPA shRNA as determined by the MAGI assay. In contrast, the number of infectious virions produced in the presence of a control shRNA targeting the HIV rev transcript was reduced three orders of magnitude compared to the negative control. The failure of the CYPA shRNA to inhibit HIV replication and infectivity suggests that ample CYPA is already present in the cell to support efficient viral replication. To address this issue we are constructing a lentiviral vector to express the shRNA, allowing depletion of CYPA prior to HIV-1 challenge. The results of these latter experiments will also be presented.