P314: therapeutically targeting the unique barcode of mll/af4

Background: Patients harbouring MLL (KMT2A)-rearrangements with AF4 remain a high-risk subgroup in acute lymphoblastic leukaemia cases, with currently <40% event free survival in infants (<1 year). Occurring in >70% of infant cases, improved therapies are essential. The presence of the MLL/AF4 is vital for transformation and maintenance of the leukaemia, making it an ideal therapeutic target. Small interfering RNAs (siRNA) can silence disease driving genes. Fusion genes such as MLL/AF4 present with a unique sequence across the breakpoint that is only present in the cancer cells, potentially allowing for an on-target. We therefore designed an siRNA targeting the fusion site within MLL/AF4. Since siRNA alone has been shown to have poor pharmacokinetic (PK) and pharmacodynamic (PD) properties, we aimed to improve efficacy chemically modifying siRNA and encapsulating them in lipid nanoparticles. Aims: The aim of this study is to develop a lipid nanoparticle delivery system to encapsulate a therapeutic siRNA targeting the MLL/AF4 breakpoint and explore the impact of this in t(4;11) cell lines. Methods: Cells expressing the MLL/AF4 fusion were treated with an MLL/AF4 (siMA6) or control (siMM) siRNA through electroporation or encapsulation by various LNP formulations. Cellular uptake and on target efficiency were investigated in tissue culture using the t(4;11)-positive cell line SEM. Results:: Electroporations were used to optimise chemically modified siRNA for improving PK and PD of siMA6, this data suggested that >200 nM was required to achieve 60% knockdown (KD) within 24 hours. Following this we tested to reduced concentrations of siRNA to investigate if LNPs improve delivery and efficacy. Cells were given 2ug/ml of siMA6 encapsulated in either cholesterol and PEG rich LNP (LNP-chol) or sphingomyelin rich LNP (LNP-SM) and compared. Uptake of LNP-SM is slower compared to LNP-chol within the first 3 hours, 60% and 90% retrospectively. However, by 6 hours, only a 5% difference with LNP-SM achieving 90% compared to 95% in LNP-chol. Surprisingly, functional studies suggest that consequential effects are enhanced in LNP-SM. Sequentially dosed cells with LNP-chol shows a knockdown ranging from 25%-65% in both MLL/AF4 and downstream target genes when compared to the control over 9 days of sequential dosing. A 10-15% reduction in proliferation and viability was seen at day 9. When treated with LNP-SM these consequences were enhanced, KD of MLL/AF4 is reduced 40-60% and downstream genes such as PROM1 and ANGPT1 to >70% by day 9. Furthermore, proliferation of the cells is impaired, reflected in substantial decrease in viable cells by 1.5-fold and notable increase was observed in the G1 and subg1 populations of the cells cycle. Surprisingly, both LNP-chol and LNP SM exhibited a strong impact on self-renewal with a reduction of ≥50% and ≥60% retrospectively by day 9. Summary/Conclusion: This study has demonstrated the advantage of targeting fusion genes with a therapeutic siRNA. We further prove the impact of using an LNP delivery system to improve therapeutic potential of siRNA. This allowed use to more clinically relevant concentrations to achieve a 60% of MLL/AF4 and downstream target genes. Notably, using an LNP-SM formulation enhanced activity of siMA6, showing a substantial impact on cell proliferation, viability, and capacity to self-renew. The next important step of this project is to test PK properties in-vivo and validate the promising findings observed in-vitro. These encouraging results could lead to improved treatments for these ALL patients with a clinically poor prognosis