Please use this identifier to cite or link to this item: http://theses.ncl.ac.uk/jspui/handle/10443/4806
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dc.contributor.authorIssa, Hasan-
dc.date.accessioned2020-11-10T15:36:48Z-
dc.date.available2020-11-10T15:36:48Z-
dc.date.issued2019-
dc.identifier.urihttp://theses.ncl.ac.uk/jspui/handle/10443/4806-
dc.descriptionPhD Thesisen_US
dc.description.abstractThe translocation t(8;21) stands out as a paradigm of genomic aberrations in which it’s fusion product (RUNX1/ETO) initiates and maintains the leukaemogenic transformation of haematopoietic stem cells. Like all leukaemias, t(8;21)-positive AML is characterised by extensive hyperproliferation and aberrant self-renewal. Here, in this thesis we show that silencing RUNX1/ETO by siRNA dysregulates the leukaemic molecular programme, restrains leukaemia expansion and ablates clonogenicity, suggesting therapeutic potentials. Aiming to develop a specific targeted therapy, we relied on the RNAi machinery to specifically knockdown RUNX1/ETO by a chemically modified siRNA encapsulated into a lipid nanoparticle. To enhance the efficacy of siRUNX1/ETO, we have introduced a combination of 2’sugar and phosphodiester backbone modifications and proved the robustness of the gene knockdown in vitro. Then, we have utilised a state-of-the-art microfluidic system to encapsulate the siRNA into a novel lipid nanoparticle that is approved for clinical use. The lipid nanoparticles provided a long-lasting RUNX1/ETO knockdown in vitro in cell lines, and in t(8;21) patient primary cells and PDXs in a co-culture system. The gene knockdown was also associated with irreversible changes in RUNX1/ETO transcriptional network and induced a cytostatic phenotype characterised by G1 cell cycle arrest and senescence. To gain insight into the pharmacokinetics and biodistribution of the nanoparticles, we have developed a protocol to label the lipid nanoparticles with a NIR dye that is compatible with in vivo imaging. Systemic administration of a single dose of the labelled nanoparticles caused a global body distribution in immunocompromised hosts, including leukaemic tissues and CNS. Our in-house optimised labelling protocol transforms the nanoparticles into a platform for tissue-specific delivery by substituting the dye with targeting motifs. Using comprehensive experimental settings in vivo, we have successfully achieved RUNX1/ETO knockdown in t(8;21) murine xenotransplantation model and proved the on-target effect of the nanoparticles. Further ex vivo testing revealed that II RUNX1/ETO repression triggers cellular senescence and initiates a myeloid differentiation programme, which were affirmed by RNA-seq analysis obtained from RUNX1/ETO knockdown mice. Our study, for the first time, shows that RUNX1/ETO depletion in vivo in a clinically relevant setting significantly prolongs the survival of leukaemic mice. Our novel approach for gene knockdown revealed that depleting RUNX1/ETO severely impairs clonogenicity and self-renewal, as well as prevents leukaemia propagation in secondary recipients. To exploit the therapeutic benefits of combinational chemotherapies with RUNX1/ETO knockdown, we carried in vitro drug toxicity assays and in vivo treatment. The combination of RUNX1/ETO depletion with DNA damage agents treatment resulted in a lost sensitivity to those chemotherapies. On the contrary, RUNX1/ETO knockdown enhanced the pharmacological inhibition of BCL2 with a small molecule inhibitor in vitro but not in vivo. Overall, this thesis developed a potent siRNA-based drug for RUNX1/ETO knockdown, which brings gene therapy from the lab closer to bedside. The thesis supports the notion that RUNX1/ETO guards the reservoir of leukaemic initiating cells, and shows how targeting the fusion transcript with siRNA significantly reduces the number of the long-term repopulating leukaemic stem cells providing an effective second-line therapy to prevent relapse in AML.en_US
dc.description.sponsorshipAKF-ISP, AKF-EB-UKen_US
dc.language.isoenen_US
dc.publisherNewcastle Universityen_US
dc.titleTherapeutic targeting of the leukaemic fusion genesen_US
dc.typeThesisen_US
Appears in Collections:Northern Institute for Cancer Research

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