Antisense approaches towards HIV-1

Several forms of therapeutic antisense molecule have been developed. These include small interfering RNAs (siRNAs), short hairpin (shRNAs) and longer single stranded antisense RNAs. In this study, steric blocking RNA oligonucleotides (ONs) and their analogues were investigated. The packaging process of HIV-1 is highly specific and involves an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. This region is known as Ψ and comprises four stem loops (SL1-4), within which SL3 is a high affinity binding site for Gag. Deletion of SL3 severely reduces viral packaging. High affinity ONs targeted to Ψ inhibited Gag binding in vitro and confirmed SL3 as the major packaging determinant SL2 was also shown to influence Gag binding, but to a lesser extent. HIV-1 replication was inhibited following cellular delivery of SL3 targeted ONs. An ON targeted to trans-activating response region (TAR) of HIV-1 also showed antiviral activity in these assays. From those tested, mixmer ONs consisting of 2’-O-Methyl and locked nucleic acid analogues (2’Ome/LNA) showed significant and sequence-specific inhibition of viral replication. Having identified target sequences, viral vectors were developed to deliver expressed forms of these in vivo. A minimal lentiviral vector based on HIV-2 was produced with reduced homology to packaging constructs. This demonstrated efficient gene transfer.EThOS - Electronic Theses Online ServiceGBUnited Kingdo