Antisense oligonucleotide (ASO) gene inhibition and treatment

Abstract

Embodiments of the invention related generally to antisense oligonucleotides (ASOs) and, more particularly, to compositions and methods for regulating protein synthesis using ASOs. In one embodiment, the invention provides a method of treating a patient diagnosed with a myeloproliferative disorder (MPD) comprising: administering to the patient an amount of an antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding JAK2 (ASO-T-JAK2) compound effective to treat such disorder.

Description

[0001] Antisense Oligonucleotide (ASO) Gene Inhibition and Treatment

[0002] Cross-Reference to Related Applications

[0003] This application claims priority to co-pending US Patent Application Serial No. 63 / 500,315, filed 5 May 2023, which is hereby incorporated herein as though fully set forth.

[0004] Sequence Listing

[0005] The sequence listing contained in the electronic file titled “VAND-0258- PCT_sequence_listing.xml,” created 28 April 2024 and comprising 4.79 kb, is hereby incorporated herein.

[0006] Background

[0007] Myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterized by their independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. Polycythemia vera (PV) is an acquired MPD characterized by increased blood cell mass, hematocrit count, and leucocyte count, associated with splenomegaly and incidental myelofibrosis. Most individuals diagnosed with PV carry a valine-to-phenylalanine mutation at position 617 (V617F) of the Janus Kinase 2 (JAK2) gene.

[0008] Myelodysplastic syndrome (MDS) is a myelodysplastic disorder characterized by ineffective hematopoiesis, cytopenia, and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. Both PV and MDS leave individuals at risk for progression to acute myeloid leukemia.

[0009] The JAK2 gene codes for a non-receptor tyrosine kinase involved in cell growth, development, differentiation, and histone modifications, as well as cytokine and growth factor signaling. Abnormal cytokine signaling due to an aberrant JAK2 Signal Transducer and Activator of Transcription (JAK2-STAT) pathway plays a role in the pathogenesis of both MDS and PV. JAK2 mutations are critical for oncogenesis of hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway can promote tumor cell growth, migration, proliferation, invasion, and angiogenesis. The increased kinase activity of JAK2 is present in several hematologic malignancies and the somatic JAK2 V617F gain-of-function mutation can be found in at least 95% of PV patients and in some MDS cases. An antisense oligonucleotide (ASO) is a synthetic, multi-nucleotide RNA compound that hybridizes to a target RNA sequence in a manner such that gene expression can be inhibited, including by inactivating an mRNA molecule.

[0010] Summary

[0011] In a first aspect, the invention provides a method of treating a patient diagnosed with a myeloproliferative disorder (MPD) which comprises: administering to said patient an amount of an ASO-T-JAK2 compound effective to treat such disorder.

[0012] In a second aspect, the invention provides a method of inhibiting expression of the JAK2 gene in an individual which comprises: administering to the individual an amount of an ASO-T-JAK2 compound effective to inhibit JAK2 expression in the individual.

[0013] In another aspect, the invention provides an antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding JAK2. In some embodiments, these are novel ASO-T-JAK2 compounds. Such compounds include, but are not limited to the oligonucleotide of SEQ ID 3. Numerous methods exist in the art for preparing ASOs once the nucleotide base sequence thereof have been determined. The determination of the appropriate sequence of bases for an ASO-T-JAK2 compound is likewise accomplished by strategies known in the art for identifying gene sequences for which an ASO would inhibit protein expression by the gene upon RNA binding.

[0014] In the treatment of an individual patient by administration of an ASO-T-JAK2, the dose and dosage regiment can be readily determined. Numerous examples of approved dosing for ASOs are known in the art, including nusinersen (marketed as Spinraza), mipomersen (marketed as Kynamro), and fomivirsen (marketed as Vitravene). In certain circumstances, it may be advantageous to administer the ASO- T-JAK2 in a derivatized or modified form in order to optimize bio availability, duration of action, or therapeutic effect otherwise. Methods are known in the art for such modification / derivatization. See, generally, Drug Delivery Trends in Clinical Trails and Translational Medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics, J Pharm Sci. (2011 Jan); 100(1): 38-52. For the purposes of the present invention the ASOs are administered intravenously, though other routes of administration are possible, including oral administration. Such alternative routes of administration are applicable to the ASOs described herein.

[0015] Accordingly, a further aspect of the present invention includes pharmaceutical compositions that are used for the administration of the ASO-T-JAK2 compound to the patient being treated. These include sterile parenteral dosage forms of the type conventionally employed for the intravenous administration of a medicine. Such pharmaceutical compositions contain excipients that may be required. These can include one or more diluents, carriers, adjuvants, or combinations thereof as are known in the art for the production of a finished dosage form.

[0016] Detailed Description

[0017] As used herein, the term “ASO-T-JAK2” refers to an ASO targeted to a nucleic acid molecule encoding the gene JAK2. The term “nucleic acid molecule” is a reference to a polynucleotide compound, e.g., RNA.

[0018] An illustrative ASO-T-JAK2 compound is disclosed herein as including SEQ ID No. 3, which is complementary to a target sequence (SEQ ID NO. 2) in the JAK2 gene. Additional ASO-T-JAK2 compounds can be identified and prepared by methods known in the art for developing antisense oligonucleotides specific to a target RNA sequence.

[0019] The invention provides a method of treating a patient diagnosed with an MPD, which may be, for example, PV or MDS, which comprises administering to the patient an amount of an ASO-T-JAK2 compound effective to treat the MPD. An amount of the ASO-T-JAK2 compound effective to treat MPD requires administration of the compound in a quantity effective to inhibit JAK2 protein expression in the patient.

[0020] A “patient” as referenced above references an individual diagnosed with, suffering from, or at risk of developing, being diagnosed with, or suffering from a disease or disorder, specifically an MPD such as PV or MDS.

[0021] Embodiments of the invention provide target sequences in the JAK2 pre-mRNA that are amenable to binding by a synthetic ASO. When so bound, expression of the JAK2 gene is inhibited. In patients diagnosed with an MPD, such as PV or MDS, such inhibition of JAK2 expression provides an effective method of treatment of the disorder.

[0022] As noted above, PV and MDS are both associated with the V617F mutation of the JAK2 protein. The wildtype JAK2 protein sequence is shown in SEQ ID NO 1 in which the amino acid at position 617 is valine.

[0023] In one aspect of the present invention, a target region within the JAK2 pre-mRNA molecule is identified that, when bound with an ASO, is capable of inhibiting synthesis of the JAK2 protein. This region includes the 19 bp sequence shown in SEQ ID NO 2.

[0024] ATAGTTCATAATCTGGAGA (SEQ ID NO 2) The complementary ASO sequence is shown in SEQ ID NO 3.

[0025] 5’-UCUCCAGAUUAUGAACUAU-3’ (SEQ ID NO 3)

[0026] SEQ ID NO 3 bridges an intron and exon of the JAK2 pre-mRNA. When ASOs containing this sequence or portions of this sequence are bound to JAK2 pre-mRNA, processing of the pre-mRNA into mature mRNA is prevented, as is synthesis of the JAK2 protein.

[0027] To test the efficacy of ASOs having SEQ ID NO 3 to bind to the JAK2 pre-mRNA and inhibit JAK2 protein synthesis, a HEL cell line including the V617F gain-of- function variant noted above is treated with a 19mer ASO of SEQ ID NO 3 (1 pM) for 72 hours. The ASO is designed with a phosphorothioate 2’-0-methoxyethyl (MOE) backbone and prioritized based on in silico binding affinity and limited off- target binding. Flow cytometry and fluorescein labeling are used to confirm cellular entry.

[0028] Results show a significant decrease (approximately 50%) in JAK2 protein levels in ASO-treated samples compared to control (untreated) samples. JAK2 qPCR results confirm 40-60% of target transcripts. STAT5 phosphorylation status further confirms this effect, with a 35% pSTAT5 reduction.

[0029] The ASO of SEQ ID NO 3 shows limited off-target effects in silico. Overall, this ASO is a highly specific JAK2 agent affecting direct levels of JAK2 protein synthesis as well as downstream STAT signaling.

[0030] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0031] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any related or incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

CLAIMS1. A method of treating a patient diagnosed with a myeloproliferative disorder (MPD) which comprises: administering to said patient an amount of an ASO-T-JAK2 compound effective to treat such disorder.

2. The method of claim 1, wherein the ASO-T-JAK2 compound is targeted to SEQ ID NO 2.

3. The method of claim 2, wherein the ASO-T-JAK2 compound comprises a nucleotide sequence including SEQ ID NO 3.

4. The method of claim 1, wherein the amount of said ASO-T-JAK2 compound is an amount sufficient to inhibit expression of JAK2 in the patient.

5. The method of claim 1, wherein the MPD is selected from a group consisting of: polycythemia vera (PV) and myelodysplastic syndrome (MDS).

6. A method of inhibiting expression of the JAK2 gene in an individual which comprises: administering to the individual an amount of an ASO-T-JAK2 compound effective to inhibit JAK2 expression in the individual.

7. The method of claim 6, wherein the ASO-T-JAK2 compound is targeted to SEQ ID NO 2.

8. The method of claim 7, wherein the ASO-T-JAK2 compound comprises a nucleotide sequence including SEQ ID NO 3.

9. An antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding JAK2.

10. The ASO of claim 9, wherein the ASO is targeted to SEQ ID NO 2.

11. The ASO of claim 10, wherein the ASO comprises a nucleotide sequence including SEQ ID No 3.

12. A pharmaceutical composition which comprises: the ASO of claim 9; and a pharmaceutically acceptable diluent, carrier, adjuvant, or combination thereof.

13. The pharmaceutical composition of claim 12 which further comprises a sterile parenteral dosage form.

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