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The Therapeutic potential of lentivector-delivered RNAi

Casey, NP 2010 , 'The Therapeutic potential of lentivector-delivered RNAi', PhD thesis, University of Tasmania.

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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.

Item Type: Thesis - PhD
Authors/Creators:Casey, NP
Keywords: lentivirus, vector, RNAi, shRNA, chrosomal-translocation, leukaemia
Additional Information:

Copyright 2010 the author - I certify that this thesis contains no material which has been accepted for a
degree or diploma by the University or any other institution, and that to the best of my
knowledge and belief, no material previously published or written by another person has
been utilised in the preparation of this thesis, except where due acknowledgement is
made in the text, nor does this thesis contain any material which infringes copyright.

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