Paper Details

ABSTRACT

Background 
Polycythemia vera (PV) is a clonal myeloproliferative neoplasm driven by a somatic JAK2-V617F point mutation (c.G1849T) in ~95% of patients, causing erythrocytosis, hyperviscosity, and thrombotic risk. JAK2 inhibitors such as ruxolitinib lack allele selectivity, also impair wild-type (WT) JAK2-dependent hematopoiesis leading to various cytopenias, and do not reduce mutant clonal burden. Pegylated interferon alphas produce slow, incomplete molecular responses constrained by toxicity. Allele-specific JAK2-V617F knockdown is potentially disease-modifying by reducing mutant allele burden while sparing WT hematopoiesis of long-term hematopoietic stem cells (LT-HSC). 
XNAzymes are chemically modified short DNA sequences composed of two RNA binding arms flanking an enzymatic core that catalyzes purine-pyrimidine RNA cleavage independently of cellular cofactors. The 5′ arm of the JAK2V617F targeting XNAzyme, binds via Watson-Crick complementarity to the mutant transcript and supports RNA cleavage; a single mismatch against the WT transcript confers selectivity and chemical modifications ensure metabolic stability for sustained in vivo activity. 

Methods 
1EXN783, a NOV340-formulated XNAzyme targeting human JAK2-V617F, was administered IV to lethally irradiated C57BL/6 recipients of bone marrow from SclCre;JAK2-V617F;GFP donors. Treatment was initiated 4 (moderate disease; ~60% peripheral blood RBC chimerism, 8 weeks) or 7 weeks after transplant (severe disease; ~90% peripheral blood RBC chimerism, 5 weeks); Ruxolitinib (60 mg/kg PO BID) served as comparator. Efficacy was assessed longitudinally by peripheral blood hematologic parameters, including hematocrit, hemoglobin, RBC counts, platelet counts, leukocyte subsets, and peripheral JAK2-V617F chimerism. At termination, BM and spleen were analyzed by flow cytometry to quantify JAK2-V617F chimerism across hematopoietic stem cells and relevant downstream progenitor compartments. Target engagement was assessed by RT-qPCR for hJAK2-V617F in sorted CFU-E and pre-CFU-E populations from BM and spleen. LT-HSC functional activity and disease-propagating capacity were assessed by secondary transplantation of BM from treated donors into lethally irradiated recipients followed for 12 weeks without further treatment. 

Results 
Biodistribution and target engagement: 1EXN783 reached BM and splenic hematopoietic compartments in a dose-dependent manner, with uptake exceeding 90% in LT-HSCs at 15 mg/kg and comparable uptake across CFU-E, pre-CFU-E, preMegE, pre-GM, and MKP progenitors. In uptake-positive CFU-E from BM and spleen, 1EXN783 reduced hJAK2-V617F mRNA by ~70% versus vehicle while completely sparing mouse WT Jak2, confirming allele selectivity.  
Primary BM Transplant Model: 1EXN783 produced a complete hematologic response, normalizing hemoglobin, hematocrit, and RBC counts into the WT range and erythroid parameters like ruxolitinib. PV reversal was further supported by a significant decrease in plasma IL-6. Critically, JAK2-V617F chimerism in BM LT-HSCs was significantly reduced, a stem-cell molecular response not seen with ruxolitinib.  
Secondary transplant: BM from 1EXN783-treated donors failed to develop PV in secondary recipient mice. Erythroid parameters stayed within normal range over 12 weeks, and peripheral JAK2-V617F chimerism declined resulting in deep molecular response (granulocyte allele burden <20% at week 12) in 75% of recipients. In 90% of 1EXN783-treated recipient mice, mutant LT-HSCs were undetectable in BM. In contrast, secondary recipients from both vehicle- and ruxolitinib treated donors sustained erythrocytosis with high peripheral blood mutant chimerism. 1EXN783 mutant LT-HSC depletion exceeded that previously reported for pegylated interferon-α in preclinical studies. 

Conclusions  
1EXN783 is the first allele-specific catalytic enzyme to selectively deplete JAK2-V617F mutant LT-HSCs in vivo and modify treated marrow cells to be incapable of re-establishing PV following secondary transplantation. The combination of complete hematologic response, deep stem-cell molecular response, and durable disease eradication supports a potential long-term disease-modifying effect. These data differentiate 1EXN783 from JAK2 inhibitors and interferon-based approaches and establish XNAzymes as a best-in indication platform for the treatment of V617F+ MPNs.