Development of a JAK2V617F-targeting lipid nanoparticle-siRNA as a novel gene therapy for myeloproliferative neoplasms

Abstract Background A single acquired JAK2 point mutation (JAK2V617F) drives myeloproliferative neoplasm (MPN) disease in 50-60% of essential thrombocythemia (ET) and myelofibrosis (MF) patients and up to 95% of polycythemia vera (PV) patients. Current treatment relies on life-long use of JAK inhibitors, in particular ruxolitinib, which provides symptomatic relief and splenomegaly reversal for most patients but does not selectively target or kill mutant clones. Over 50% of patients discontinue ruxolitinib within 3 years due to severe side-effects. Lipid Nanoparticles (LNPs) are the leading non-viral delivery vehicles which can encapsulate nucleic acids, providing a safe, low toxicity and effective platform to deliver RNA gene-therapy to cells both in vitro and in vivo. Here, we present pre-clinical data for W-0301, a novel JAK2V617F-specific LNP-siRNA therapeutic for the treatment of MPNs. Methods We rationally designed novel LNP formulations to enhance nucleic acid delivery to the bone marrow. Successful delivery was determined by assessing mCherry fluorescence in bone marrow cells by flow cytometry following IV injection of LNP encapsulating mCherry mRNA. Target knockdown in a panel of MPN cancer cell lines was assessed by qRT-PCR and western blot. Killing efficacy was assessed by flow cytometry. In vivo target knockdown was assessed by scRNAseq. To test the efficacy of W-0301 to target mutant HSPCs we performed CFU assays with ET and PV patient PBMCs. To gain further pre-clinical data W-0301 was administered in vivo to mice harbouring the JAK2V617F mutation and disease phenotype in blood, bone marrow and spleen was assessed by flow cytometry, histology and scRNAseq. Results We formulated next-generation LNPs to efficiently target HSPCs in bone marrow via IV injection without the need for HSPC-targeting ligands such as antibody binders. Following a single IV injection of ligand-free LNP containing an mCherry mRNA, up to 20% of LTHSCs and MPPs were positive for mCherry after 24h, indicating the functional delivery of the nucleic acid cargo via the LNP delivery system; this signal was retained for at least 72h. A panel of LNP-siRNAs directed against JAK2V617F was screened and the most promising candidate, W-01, was carried forward for further evaluation of therapeutic potential. Encapsulation of W-01 within the optimal LNP (.03) created the LNP-siJAK2V617F therapeutic, W-0301. W-0301 demonstrated highly selective and dose-dependent knock down of JAK2V617F, but not JAK2WT, when added to the culture media of a panel of MPN cell lines. This resulted in highly efficient cell killing of JAK2V617F-positive cells via apoptosis. Ability of PBMCs from JAK2V617F+ PV patients to grow BFU-E and CFU-GM colonies was significantly reduced, indicating either depletion of HSPCs or loss of proliferation potential as a result of W-0301 treatment. In a transgenic model of JAK2V617F homozygosity, in which mice develop a PV-like phenotype, scRNAseq revealed that IV delivery of W-0301 resulted in knockdown of JAK2V617F mRNA in HSPCs and induced cell death within the bone marrow compared to a non-targeting LNP-siRNA control. Finally, JAK2V617F homozygous mice were administered repeat doses of W-0301 over 2 weeks. Mice displayed no weight loss or adverse symptoms, indicating that the therapy was well tolerated. Within the bone marrow, we observed a dramatic reduction in CD71+ disease-driving erythroid progenitor cells and more mature reticulocytes, to levels comparable with JAK2WT littermate controls. Conclusions We present here a novel therapeutic W-0301 for JAK2V617F+ MPNs, combining the mutation-specific nature of RNAi with a rationally designed, ligand-independent LNP platform for delivery of gene therapy to bone marrow. W-0301 induced JAK2V617F-specific gene-silencing, followed by mutation-specific cell killing of JAK2V617F HSPCs in vitro and in vivo, and amelioration of key disease pathologies. Our study offers a new approach for JAK2V617F mutant–selective inhibition through apoptosis-induced eradication of mutant HSPCs, therebyrepresenting a potential curative strategy for patients with JAK2V617F positive MPNs. It furthermore demonstrates the potency of our targeting ligand-independent LNP-RNA platform technology to create therapeutics for other haematological malignancies with high driver-mutation prevalence.