Delivery of a Therapeutic siRNA Targeting the MLL/AF4 Fusion Gene

Abstract

The translocation between chromosomes 4 and 11 generating the KMT2A/AFF1 (will be referred to as MLL/AF4) fusion gene remains one of the most prevalent cytogenetic subtypes in infant (<1 yr) acute lymphoblastic leukaemia (ALL) cases. Despite substantial improvements in childhood ALL over the past 5 decades, these patients remain in the high-risk category with only <40% chance of event free survival. This project aimed to develop a lipid nanoparticle (LNP)-based siRNA delivery approach to specifically silence the MLL/AF4 fusion gene both in vitro and ex vivo. A secondary aim was the optimisation of the ex vivo culture of primary leukaemic cells from patients with t(4;11) ALL. To enhance the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the siRNA, we introduced several types of 2’-modifications in combination with terminal phosphorothioates and compared their knockdown efficacy and phenotypic consequences of siRNA transfection in t(4;11) cell lines SEM and E-SEM and patient derived material. Delivery by electroporation, both unmodified and modified siRNA designs exhibited similar gene expression knockdown of MLL/AF4 and concomitantly reduced downstream targets including HOXA7, PROM1 and ANGPT1. All siRNAs displayed specificity to the MLL/AF4 fusion, with minimal effects observed in a t(8;21) cell line. Packaging into LNPs improved the PD properties by reducing the required concentration 3-fold to achieve a similar impact on gene expression as the electroporation. Furthermore, an increased functional effect was observed in cells treated with the fully modified design. Encapsulation into a sphingomyelin rich LNP further increased impairment on leukaemic potential. The use of a targeted LNP did not enhance any effects despite accelerated uptake kinetics compared to the untargeted LNP. Both untargeted and targeted LNP formulations failed to achieve significant MLL/AF4 knockdown in primary or patient-derived xenograft ALL cells, while electroporation yielded a 50% reduction of MLL/AF4. These data suggest on target efficacy, however, LNP delivery requires further optimisation. To enable the mid- and long-term ex vivo culture of patient derived samples I developed a co culture system with mesenchymal stromal cells (MSCs) and a tailored media with several cytokines. This set-up promoted proliferation of CD19+CD133+ ALL blasts, while conserving their immunophenotype and MLL/AF4 expression over the culturing period. These conditions also supported a likely healthy myeloid cell with limited proliferative capacity. Upon exhaustion and cell death, the ALL cells continued to proliferate for >7 weeks. This co-culture platform will facilitate pre-clinical drug testing ex vivo in MLL/AF4+ samples.