The Nanoparticle-Mediated Delivery of Therapeutic Sirnas Targeting the KMT2A::Afdn Fusion Gene in AML
Authors: du Plessis Manisha , Mata Casimiro Luis Daniel , Jeremias Irmela , Heidenreich Olaf 
Published: December 2024

Acute myeloid leukemia (AML) is characterized by the presence of chromosomal rearrangements that result in novel fusion-oncoproteins. Since fusion genes both initiate and maintain leukemia, they are cancer-specific therapeutic targets that are expressed in every single leukemic cell. We have developed a therapeutic siRNA approach by designing lipid nanoparticles (LNPs) for the in vivo delivery of siRNAs to AML cells (Issa et al, 2023; Swart et al, 2023). We have now extended this approach to t(6;11)-positive AML that is associated with a dismal outcome in pediatric and adult AML. This translocation fuses the KMT2A (MLL) gene with the AFDN (AF6) gene, resulting in the dysregulation of the RAS pathway, aberrant gene expression and impaired differentiation.
Here, we tested both an unmodified and a fully modified, 21-23 nucleotide, overhang siRNAs that successfully targets the KMT2A::AFDN gene fusion in AML cell lines and t(6;11) primary cells. Four different LNP formulations were tested, with changes both in lipid composition and molar ratios, to optimize knockdown efficiency. First, we assessed the delivery and knockdown efficiency using the Onpattro (Patisiran) LNP formulation, which consists of DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 at the molar ratios 50:10:38.5:1.5, respectively. Second, we utilized a LNP formulation where the cholesterol helper lipid was substituted with a naturally occurring phytosterol, namely β-sitosterol, at the same molar ratio as the standard formulation. Third, we tested an LNP formulation consisting of DLin-MC3-DMA, cholesterol, sphingomyelin and DSPC with molar ratios of 50:20:20:10. Lastly, we also tested a novel LNP formulation, which incorporates DLin-MC3-DMA, β-sitosterol, sphingomyelin and polysarcosine.
Here, a 85% and 82% knockdown for the standard formulation, a 74% and 80% knockdown for the β-sitosterol formulation, a 76% and a 77% knockdown for the sphingomyelin formulation and a 83% and a 76% knockdown for the novel formulation were observed at a concentration of 2 µg/ml in SHI-1 cells over 48 hours when comparing the unmodified and modified siRNAs, respectively. These data indicates that knockdown efficacies are similar, while the fully modified siRNA only confers marginal increases in knockdown efficiency. As such, the choice of the optimal LNP for the treatment of AML will then depend on the pharmacokinetics and the ability of the LNPs to accumulate in the bone marrow in vivo.
Additionally, the formation of the biomolecular protein corona upon serum exposure is an important factor mediating LNP efficiency. Serum proteins adsorbed to LNPs can facilitate LNP uptake and endosomal escape. We assessed both the association of LNPs encapsulating a cy5-labelled siRNA with SHI-1 and ML-2 cells in serum and in serum-free conditions using flow cytometry, and the knockdown efficiency. Both uptake of LNPs and knockdown were severely diminished over time in serum free conditions, and only 20% knockdown of KMT2A::AFDN was observed, suggesting that the protein corona facilitates LNP mode of action.
To investigate whether our LNP-based siRNA delivery system targeting KMT2A::AFDN confers a functional effect on downstream targets, we assessed the expression of SHARP1. SHARP1 binds to transcriptionally active chromatin and activates gene expression critical for cell survival. Furthermore, SHARP1 cooperates with KMT2A::AFDN to regulate gene expression crucial for leukemogenesis, and mediates colony formation. Indeed, we report a 50% knockdown of SHARP1 in response to KMT2A::AFDN knockdown, while observing a 2-fold decrease in colony formation of SHI-1 cells. In addition, we assessed the expression of surface markers. Here, the expression of CD117 was decreased following KMT2A::AFDN knockdown, which indicates a shift to a more mature phenotype.
Taken together, we successfully induced knockdown of KMT2A::AFDN in AML using different formulations of LNPs, a process which is mediated by the protein corona. We also gain insights into the downstream targeting of KMT2A::AFDN, where SHARP1 is downregulated upon KMT2A::AFDN knockdown, which is mediated through the loss of DOT1L activity. Furthermore, the colony formation was decreased following KMT2A::AFDN knockdown, which is in line with the loss of SHARP1. As such, the knockdown of KMT2A::AFDN with LNPs shows great potential in the treatment of t(6;11) AML.