The Therapeutic potential of lentivector-delivered RNAi

Many forms of leukaemia are caused by chromosomal translocations, which result in specific and characteristic genomic sequences. Where these sequences are unique to the leukaemic cells, they represent good candidates for targeting by sequencespecific techniques, such as RNA-Interference (RNAi). RNAi is a mechanism inherent in eukaryotic cells which silences target mRNAs based on homology to a dsRNA template. This template may be introduced artificially by a number of methods, and so this mechanism can be manipulated to regulate the expression of target genes. One of the most efficient methods of introducing RNAi templates is by expression of shorthairpin RNA (shRNA) cassettes from DNA plasmids or vectors. Lentiviral vectors are based on viruses that integrate into the DNA of the host cell, and are a highly efficient class of vectors for transducing and providing stable transgene expression in a range of cell types. They are particularly effective at tansducing haematopoietic cells, which have proven difficult to transduce by other methods. By fine-tuning the methods of vector production and transduction, a range of human leukaemic cells lines were able to be transduced with unprecedented efficiency in the present study. Lentiviral transduction was combined with rapid puromycin selection to generate a pure population of transduced cells with minimal expansion of the cell population. The strategy of expressing shRNAs from retroviral and lentiviral vectors combined with puromycin selection was used to target three well-characterised fusion genes; Bcr-Abl, PML/RARα and RUNX1/ETO, in three human leukaemic cell lines. In two of these, the shRNA was able to efficiently and effectively down-regulate the target mRNA, and inhibit the proliferation of the transduced leukaemic cells. In the third case, RUNX1/ETO, no effective shRNA design could be identified. Finally, concerns over the safety of integration targeting by current gene therapy vectors motivated an investigation of the activity of a novel integrase enzyme from the Ty3 retrotransposon found in yeast. In yeast cells, the integration-mediating enzyme of this retrotransposon has very specific targeting characteristcs, which, if retained in human cells, would provide a very safe gene therapy vector. It was found that this enzyme is indeed active in human cells, and therefore has potential in the context of human gene therapy