Entrevistas

As irregular cell shape can indicate entry of Kasumi cells into the differentiation pathway, regular observations of cellular appearance were made by light microscopy. From the first observation on the day of treatment up until the last time points examined (ie. 144 hours), all cells treated with the shRNAs remained regular in shape, indicating that none of the shRNAs were inducing differentiation.

6.4 Discussion

The RUNX1-ETO fusion gene found in Kasumi cells is responsible for many cases of acute myeloid leukaemia. Hammerhead ribozymes (Kozu et al. 2000; Szyrach et al. 2001) and siRNAs (Heidenreich et al. 2003) have been used to down-regulate the fusion mRNA, resulting in down-regulation of the protein, and impaired clonogenicity of Kasumi cells, although effects on cell proliferation were somewhat delayed (Martinez et al. 2004). Effective delivery of these molecules, however, has so far been inefficient, and one aim of this chapter was to enhance the delivery efficiency of effective gene therapy using lentiviral vectors. Transduction of Kasumi cells has in the past proved a challenge to delivery of therapeutics in vitro.

As has been outlined in Chapter 3, the modified lentiviral vectors and transduction techniques developed and used in this thesis reliably achieved high rates of transduction in Kasumi cells. To date there have been few if any reports of retroviral transduction of Kasumi cells, and this is the first report of successful transduction of these cells with lentiviral vectors. This strategy, combined with optional positive selection of transduced cells (see Chapter 3), provides a basis for effective and stable gene therapy of Kasumi cells and similar cell types. These results open the way for further investigations using compatible methodologies, such as hammerhead ribozymes and RNAi. The implications of these results extend beyond this, however, as many primary or patient-derived cell types have proven difficult to transduce with existing vector types, including lentiviral vectors. The methods and vectors developed here may therefore have application across a wide range of gene therapy protocols. This is discussed further in the General Discussion (Chapter 8).

Adapting RNAi molecules to delivery by lentiviral vectors required the identification of an shRNA molecule that was effective against the RUNX1-ETO fusion of Kasumi cells. A series of three different shRNAs molecules was designed using siRNA design algorithms, then cloned into lentiviral vectors for delivery and expression. Of the three shRNAs tested here, one bore homology with the effective siRNA design of Heidenreich et al. (2003), while the other two were novel designs. To test these designs, the shRNAs were delivered by lentiviral vectors, and the effects on levels of the RUNX1-ETO mRNA assessed by qRT-PCR.

The β-2-microglobulin house-keeping gene was used for normalisation as it was found to be one of the most stable in a comparison of commonly used house-keeping genes (Schmittgen and Zakrajsek 2000). When RUNX1-ETO mRNA levels were

normalised to β-2-microglobulin, however, there was high variability between replicates. Consequently, samples were reanalysed and normalised against another commonly used house-keeping gene, GAPDH. This gene was selected as it was used previously to study RNAi against RUNX1-ETO (Heidenreich et al. 2003), however results normalised to GAPDH were also highly variable. Therefore the qRT-PCR results provided no evidence of a down-regulatory effect by any of the three shRNAs on RUNX1-ETO mRNA levels.

Despite widespread use as a house-keeping gene, GAPDH has been reported to be susceptible to perturbation by a range of factors (Schmittgen and Zakrajsek 2000), and qRT-PCR studies of RUNX1-ETO levels in Kasumi cells have previously proved unreliable when normalised against GAPDH (Krauter et al. 1999). While there have been no such reports in relation to β-2-microglobulin, problems with quantification of mRNA in the Kasumi cell line are frequently encountered (A. Holloway, pers. comm.). As such, future studies with this cell line must start with the identification of reliable housekeeping genes. One candidate housekeeping gene that has proven reliable after stringent testing is c-Abl (Beillard et al. 2003), and it may be worth examining the reliability of this housekeeping gene in Kasumi cells.

As the qRT-PCR results were unreliable, cells were examined for likely downstream effects of down-regulation of the RUNX-/ETO fusion mRNA, specifically induction of cell differentiation and inhibition of cell proliferation. Specific targeting and down-regulation of the RUNX1-ETO mRNA by catalytic ribozymes and antisense RNA results in rapid inhibition of proliferation, with changes in viable cell number evident within 48 hours (Kozu et al. 2000). Considering the expected delay in shRNA expression following vector delivery, changes in viable cell number might therefore be expected as soon as 72 or 96 hours. Despite examination of cell numbers from 72 hours to 144 hours, however, there was no evidence of any inhibitory effect of the shRNAs on Kasumi cell proliferation. Similarly there was no evidence of differentiation in the transduced cell populations. It should be noted, however, that when RUNX1-ETO mRNA was targeted by siRNAs, the inhibitory effect was delayed by comparison with

the ribozyme and antisense methods. Whereas the latter methods both inhibit cell proliferation within 48 hours, a single treatment with siRNA did not affect cell proliferation within 5 days, despite effective and sustained down-regulation of RUNX1- ETO mRNA and protein over this period (Heidenreich et al. 2003; Martinez et al. 2004). Instead, repeated treatments with siRNA were necessary to bring about changes to cell number, which was not evident until approximately day 5 (Martinez et al. 2004). Results from this thesis have demonstrated that despite effective transduction and knockdown of the fusion mRNA and proteins, a subpopulation of cells, that has received a lower effective dose of shRNA, may survive and lead to recovery of the transduced population (see Chapters 4, 5). This recovery was typically observed within a matter of days. Considering the delayed effect of the siRNAs on cell proliferation, it was therefore unlikely that any inhibitory effect of the shRNAs delivered by the lentiviral vectors would be observed before possible recovery of the transduced cell population.

6.4.1 Conclusion

From the results presented in this chapter it must be concluded that the three shRNAs designed to target the RUNX1-ETO mRNA were ineffective in down- regulating this target. This was despite the close homology of one of these designs with an effective siRNA design. Furthermore, it should be noted that an shRNA design based on this active siRNA has elsewhere been reported to successfully down-regulate the RUNX1-ETO mRNA target (Fazi et al. 2007). The design of this active shRNA has not been published, however, and a comparison with the shRNA designs used here has not been possible. Nevertheless the results from this chapter highlight the differences between optimal siRNA and shRNA designs, and emphasise the need for design algorithms more suitable for shRNA design.