Antisense oligonucleotide (ASO) gene inhibition and therapy

Abstract

Embodiments of the present invention relate generally to antisense oligonucleotides (ASOs), and more specifically to compositions and methods for controlling protein synthesis using ASOs. In one embodiment, the present invention provides a method for treating a patient diagnosed with a myeloproliferative disorder (MPD), comprising administering to the patient an effective amount of an antisense oligonucleotide (ASO) (ASO-T-JAK2) that targets a nucleic acid molecule encoding a JAK2 compound.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of co - pending U.S. Patent Application No. 63 / 500315, filed on May 5, 2023, and the entire disclosure of the U.S. Patent Application is incorporated herein as if fully set forth herein.

[0002] Sequence Listing The sequence listing contained in the electronic file named "VAND - 0258 - PCT_sequence_listing.xml" created on April 28, 2024, and having a size of 4.79 kb is incorporated herein.

Background Art

[0003] Myeloproliferative disorder (MPD) is a clonal hematopoietic stem cell malignancy characterized by the independence or hypersensitivity of hematopoietic progenitor cells to many cytokines. Polycythemia vera (PV) is an acquired MPD characterized by an increase in blood cell volume, hematocrit value, and white blood cell count, and is accompanied by splenomegaly and occasional myelofibrosis. Most patients diagnosed with PV have a mutation (V617F) from valine to phenylalanine at position 617 of the Janus kinase 2 (JAK2) gene.

[0004] Myelodysplastic syndrome (MDS) is a myelodysplastic disorder characterized by hematopoietic dysplasia, cytopenia, and the presence of morphological dysplasia of progenitor and mature myeloid blood cells. Both PV and MDS expose patients to the risk of progression to acute myeloid leukemia.

[0005] The JAK2 gene encodes a non-receptor tyrosine kinase involved in cell proliferation, development, differentiation, histone modification, and cytokine and growth factor signaling. Abnormal cytokine signaling via the JAK2-STAT (JAK2 Signal Transducer and Activator of Transcription) pathway is involved in the pathogenesis of both MDS and PV. JAK2 mutations are important in the carcinogenesis of hematological malignancies, and excessive signaling of the JAK2-STAT pathway can promote tumor cell growth, migration, proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is seen in several hematological malignancies, and somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and some MDS cases.

[0006] Antisense oligonucleotides (ASOs) are synthetic multinucleotide RNA compounds that hybridize to target RNA sequences in a manner that inhibits gene expression, such as by inactivating mRNA molecules. [Overview of the project]

[0007] In a first embodiment, the present invention provides a method for treating a patient diagnosed with a myeloproliferative disorder (MPD), comprising administering to the patient an amount of an ASO-T-JAK2 compound effective in treating the disease.

[0008] In a second embodiment, the present invention provides a method for inhibiting the expression of the JAK2 gene in an individual, comprising administering to the individual an amount of an ASO-T-JAK2 compound effective in inhibiting JAK2 expression in the individual.

[0009] In yet another embodiment, the present invention provides antisense oligonucleotides (ASOs) that target nucleic acid molecules 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 NO: 3. In the art, once the nucleotide sequence is determined, there are numerous methods for preparing ASOs. Determining the appropriate nucleotide sequence of an ASO-T-JAK2 compound is also achieved by strategies known in the art, which identify the gene sequence to which the ASO inhibits protein expression of the gene by RNA binding.

[0010] In the treatment of individual patients with ASO-T-JAK2, the dosage and administration plan can be easily determined. Numerous approved methods of ASO administration are known in the art, including nusinersen (marketed as Spinraza), mipomersen (marketed as Kynamro), and fomivirsen (marketed as Vitravene). Under certain circumstances, it may be advantageous to administer ASO-T-JAK2 in a derivatized or modified form to optimize bioavailability, duration of action, or other therapeutic effects. Such modification / derivation methods are known in the art. For general information, see 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 this invention, ASO is administered intravenously, but other routes of administration, including oral administration, are also possible. Such alternative routes of administration are applicable to the ASO described herein.

[0011] Accordingly, further aspects of the present invention include pharmaceutical compositions used for administering the ASO-T-JAK2 compound to a patient receiving treatment. These include sterile injection formulations of the type conventionally used for intravenous administration of drugs. Such pharmaceutical compositions include any necessary excipients, which include one or more diluents, carriers, adjuvants, or combinations thereof, as known to those skilled in the art for the manufacture of the final formulation. [Modes for carrying out the invention]

[0012] As used herein, the term "ASO-T-JAK2" refers to an ASO that targets the nucleic acid molecule encoding the JAK2 gene. The term "nucleic acid molecule" means a polynucleotide compound (such as RNA).

[0013] An example of an ASO-T-JAK2 compound is disclosed herein, which contains Sequence ID No. 3, complementary to the target sequence (Sequence ID No. 2) within the JAK2 gene. Further ASO-T-JAK2 compounds can be identified and prepared by methods known in the art for developing antisense oligonucleotides specific to target RNA sequences.

[0014] The present invention provides a method for treating a patient diagnosed with MPD (which may be, for example, PV or MDS), comprising administering to the patient an amount of an ASO-T-JAK2 compound effective in treating MPD. An amount of the ASO-T-JAK2 compound effective in treating MPD requires the administration of an amount of the compound effective in inhibiting JAK2 protein expression in the patient.

[0015] In the foregoing, "patient" refers to an individual who has been diagnosed with, is suffering from, is at risk of developing, is being diagnosed with, or is suffering from a disease or disorder, particularly MPD such as PV or MDS.

[0016] Embodiments of the present invention provide target sequences in JAK2 precursor mRNA that can be bound by synthetic ASO. When such binding occurs, JAK2 gene expression is inhibited. In patients diagnosed with MPDs such as PV and MDS, such inhibition of JAK2 expression provides an effective treatment method for the disease.

[0017] As mentioned above, both PV and MDS are associated with the V617F mutation in the JAK2 protein. The wild-type JAK2 protein sequence is shown in SEQ ID NO: 1, where the 617th amino acid is valine.

[0018] In one embodiment of the present invention, a target region within the JAK2 precursor mRNA molecule is identified that, upon binding to ASO, can inhibit the synthesis of the JAK2 protein. This region includes the 19 bp sequence shown in Sequence ID No. 2.

[0019] ATAGTTCATAATCTGGAGA (Sequence ID 2)

[0020] The complementary ASO sequence is shown in sequence number 3.

[0021] 5'-UCUCCAGAUUAUGAACUAU-3'(Sequence ID 3)

[0022] Sequence ID 3 spans the introns and exons of the JAK2 precursor mRNA. When an ASO containing this sequence or a portion thereof binds to the JAK2 precursor mRNA, the processing of the precursor mRNA into mature mRNA is inhibited, and the synthesis of the JAK2 protein is also inhibited.

[0023] To verify the effectiveness of an ASO with SEQ ID NO: 3 in binding to JAK2 precursor mRNA and inhibiting JAK2 protein synthesis, the HEL cell line containing the above-mentioned V617F gain-of-function mutation is treated with a 19-base-long ASO (1 μM) of SEQ ID NO: 3 for 72 hours. The ASO is designed to have a phosphorothioate 2'-O-methoxyethyl (MOE) backbone and is preferentially selected based on in silico binding affinity and limited off-target binding. Flow cytometry and fluorescein labeling are used to confirm cellular uptake.

[0024] The results show that the JAK2 protein level is significantly decreased (by about 50%) in the ASO-treated samples compared to the control (untreated) samples. The qPCR results of JAK2 confirm 40 - 60% of the target transcripts. Furthermore, this effect is also supported by the STAT5 phosphorylation state, with a 35% decrease in pSTAT5.

[0025] The ASO of SEQ ID NO: 3 shows only a limited off-target effect in silico. Overall, this ASO is a JAK2 agent with high specificity that affects both the direct level of JAK2 protein synthesis and downstream STAT signaling.

[0026] As used herein, the singular forms of "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Also, the terms "comprises" and / or "comprising", as used herein, specify the presence of the 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.

[0027] This specification uses examples to disclose the invention, including the best mode, and to enable any person skilled in the art to practice the invention, including by manufacturing and using any device or system or by carrying out any related or incorporated method. The patentable scope of the present invention is defined by the claims and may also include other examples that a person skilled in the art could conceive. Such other examples are intended to be included in the claims if they have components that do not differ from the language of the claims, or if they have equivalent components that do not substantially differ from the language of the claims.

Claims

1. A method for treating a patient diagnosed with myeloproliferative disorder (MPD), comprising administering to the patient an amount of an ASO-T-JAK2 compound effective in treating the disease.

2. The method according to claim 1, wherein the ASO-T-JAK2 compound targets SEQ ID NO:

2.

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

3.

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

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

6. A method for inhibiting the expression of the JAK2 gene in an individual, comprising administering to the individual an amount of an ASO-T-JAK2 compound effective in inhibiting JAK2 expression in the individual.

7. The method according to claim 6, wherein the ASO-T-JAK2 compound targets SEQ ID NO:

2.

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

3.

9. Antisense oligonucleotides (ASOs) that target nucleic acid molecules encoding JAK2.

10. The ASO according to claim 9, which targets Sequence ID No.

2.

11. The ASO according to claim 10, comprising a nucleotide sequence including sequence number 3.

12. A pharmaceutical composition comprising the ASO described in claim 9 and a pharmaceutically acceptable diluent, carrier, auxiliary agent, or a combination thereof.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition further comprises a sterile injection dosage form.

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