Therapeutic targeting of the leukaemic fusion genes

PhD Thesis ; The 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 ...