Human Immunodeficiency Virus type I (HIV-1) is an RNA virus that causes Acquired Immunodeficiency Syndrome (AIDS). Gene therapy is a new treatment paradigm that aims to modify the patient s cells with therapeutic agents to reduce viral replication and disease progression. A recently discovered technology with potential application in gene therapy for HIV-1 is the RNA interference (RNAi) pathway; a natural gene suppression mechanism where a 19 21 bp dsRNA trigger prevents the translation of complementary target RNAs. The RNAi pathway can be harnessed artificially with short hairpin RNAs (shRNAs or hairpins); expressed RNA transcripts that fold into hairpin configurations by virtue of selfcomplementary regions separated by a short loop sequence. The aim of this project was to use pre-clinical models to investigate shRNA design, construction, screening and coexpression for potential use as an anti-HIV-1 therapeutic agent. A PC2-rated assay using multiple fluorescent reporters was developed to screen shRNAs for anti-HIV-1 suppressive activity, simultaneously measuring, and distinguishing, between target-specific and nonspecific shRNA activities. Phi29 DNA polymerase was used to modify the primer extension method for constructing shRNA vectors, increasing its efficiency whilst maintaining its cost effectiveness. A novel sequencing procedure using a restriction enzyme loop sequence was developed, which allowed the sequence of all shRNA vectors to be confirmed at highthroughput automated sequencing facilities. A comprehensive study of hairpin design factors showed that whilst shRNA stem length could affect processing and activity, sequence composition was the critical determinant of suppressive activity. Using the screening and construction methods developed here, many hairpins were designed and tested with at least one highly active hairpin found for each HIV-1 gene. Pre-existing and novel shRNA co-expression strategies were successfully used to co-express up to 4 hairpins to theoretically counter the emergence of viral escape mutants. Overall, this work has shown that expressed shRNAs are potentially suitable to treat HIV-1, since highly active shRNAs can be designed against all HIV-1 genes, shRNA vectors can be efficiently constructed, shRNA activities can be effectively screened and shRNAs can be combined to suppress simultaneously multiple targets.