Antisense Oligonucleotide (ASO) Gene Inhibition and Treatment

Abstract

FIELD OF THE DISCLOSURE [0002] Embodiments of the present invention relate generally to antisense oligonucleotides (ASOs), and more particularly to compositions and methods for modulating protein synthesis using ASOs. In certain embodiments, the present invention provides a method of treating a patient diagnosed with myelodysplastic syndrome (MDS), comprising administering to the patient an antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding JAK2 (ASO-T-JAK2) compound in an amount effective to treat such disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to co-pending U.S. Provisional Patent Application No. 63 / 222,336, filed July 15, 2021, and U.S. Provisional Patent Application No. 63 / 224,362, filed July 21, 2021, each of which is incorporated herein as if fully set forth. Sequence Listing The sequence listing contained in the electronic file "\txt_VAND-224-PCT.xml", created on June 19, 2022 and containing 63 kb, is incorporated herein. [Background technology]

[0002] The present invention relates to certain novel antisense oligonucleotides (ASOs), and more particularly to pharmaceutical compositions thereof and methods for treating certain diseases or conditions in which modulating protein synthesis using ASOs may have therapeutic value. More particularly, aspects of the invention relate to the treatment of known disorders such as polycythemia vera (PV) and myelodysplastic syndromes (MDS), as well as Charcot-Marie-Tooth type 2 (CMT2) disease.

[0003] PV is a myeloproliferative disorder characterized by erythroid hyperplasia, myeloid leukocytosis, thrombocytosis, and splenomegaly. Most individuals diagnosed with PV have a valine to phenylalanine mutation at position 617 of the JAK2 gene (V617F).

[0004] MDS is a group of clonal blood stem cell disorders characterized by ineffective hematopoiesis, often resulting in anemia. Some cases of MDS are associated with the same V617F mutation in the JAK2 gene, particularly in elevated platelet counts.

[0005] The JAK2 gene encodes a non-receptor tyrosine kinase that is involved in cell proliferation, development, differentiation, and histone modification, as well as cytokine and growth factor signaling.

[0006] Charcot-Marie-Tooth disease (CMT), also known as hereditary motor and sensory neuropathy, causes damage to the peripheral nerves and nerves involved in muscle control. Symptoms are progressive, often beginning in the feet and lower legs, then progressing to the fingers, hands, and arms.

[0007] Nearly all cases of CMT are inherited. More than 40 genes are associated with CMT. Some genes are associated with more than one form of CMT, and some forms of CMT are associated with more than one gene. Thus, an individual may have more than one form of CMT as a result of more than one inherited mutation. More than half of all CMT cases are caused by a duplication of the PMP22 gene on chromosome 17. The other forms of CMT are caused by X-linked mutations.

[0008] CMT2 type 2 (CMT2) is a less common subtype of CMT that affects nerve axons. Most forms of CMT2 are inherited in an autosomal dominant pattern, but some forms, including CMT2S type (CMT2S), are inherited in an autosomal recessive pattern. Thus, symptoms associated with CMT2S are typically less severe than autosomal dominant forms of CMT2. Mutations associated with CMT2S are in the IGHMBP2 (immunoglobulin μ DNA binding protein 2) gene on chromosome 11, which results in abnormal RNA processing and axonal neuropathy. Summary of the Invention [Problem to be solved by the invention]

[0009] "Antisense oligonucleotide" or "ASO" refers to a synthetic multinucleotide RNA compound that hybridizes to a target RNA sequence in such a way that gene expression can be inhibited, including inactivating an mRNA molecule. [Means for solving the problem]

[0010] The present invention provides a method for treating a patient diagnosed with MDS, comprising administering to the patient an amount of an ASO-T-JAK2 compound effective to treat MDS. According to the present invention, an ASO-T-JAK2 compound refers to any antisense oligonucleotide (ASO) that targets a nucleic acid molecule encoding JAK2. Furthermore, an amount of an ASO-T-JAK2 compound effective to treat MDS requires administration of an amount of the compound effective to inhibit JAK2 protein expression in the patient.

[0011] As stated above, the term "ASO-T-JAK2" refers to an ASO that targets a nucleic acid molecule that codes for the gene JAK2. The term "nucleic acid molecule" refers to a polynucleotide compound, i.e., RNA. "Antisense oligonucleotide" or "ASO" refers to a synthetic multinucleotide DNA compound that hybridizes to a target RNA in such a manner that gene expression can be inhibited, including inactivating mRNA compounds. Exemplary ASO-T-JAK2 compounds are disclosed herein (SEQ ID NO: 4 and SEQ ID NO: 5, which target SEQ ID NO: 2 and SEQ ID NO: 3, respectively). Additional ASO-T-JAK2 compounds can be identified and prepared by methods known in the art for developing antisense oligonucleotides specific to target RNA sequences.

[0012] The term "ASO-T-IGHMBP2" refers to an ASO that targets a nucleic acid molecule that encodes the IGHMBP2 gene. The term "nucleic acid molecule" refers to a polynucleotide compound, i.e., RNA. Exemplary ASO-T-IGHMBP2 compounds are disclosed and described in detail below. Additional ASO-T-IGHMBP2 compounds can be identified and prepared by methods known in the art for developing ASOs specific to target RNA sequences.

[0013] The "patient" referred to above refers to an individual who has been diagnosed with, is suffering from, or is at risk of developing, is suffering from, or is suffering from a disease or disorder, particularly PV or MDS or CMT2S.

[0014] In another embodiment, the invention provides a method of treating a patient diagnosed with PV comprising administering to said patient an amount of an ASO-T-JAK2 compound effective to treat PV, wherein the amount of an ASO-T-JAK2 compound effective to treat PV is an amount effective to inhibit JAK2 protein expression in the patient.

[0015] In yet another embodiment, the invention provides a method of inhibiting expression of the JAK2 gene in an individual, comprising administering to the individual an ASO-T-JAK2 compound in an amount effective to achieve such inhibition.

[0016] In yet another embodiment, the present invention provides novel ASO-T-JAK2 compounds. As mentioned above, such compounds include, but are not limited to, the oligonucleotides of SEQ ID NO: 4 and SEQ ID NO: 5. There are many methods in the art for preparing ASOs once the nucleotide sequence has been determined. Similarly, to identify the gene sequence of a gene for which the ASO inhibits protein expression by RNA binding, the determination of the appropriate sequence for the ASO-T-JAK2 compound is accomplished by strategies known in the art.

[0017] For the treatment of an individual patient with administration of ASO-T-JAK2, the dosage and dosing schedule can be readily determined. Approved administrations of ASOs are numerous and known in the art, such as nusinersen (sold as Spinraza), mipomersen (sold as Kynamro), and fomivirsen (sold as Vitravene). In certain circumstances, it may be advantageous to administer a derivatized or modified form of ASO-T-JAK2 to optimize bioavailability, duration of action, or other therapeutic effect. Methods for such modification / derivatization are known in the art. See generally, Drug Delivery Trends in Clinical Trails and Translational Medicine: challenges and opportunities in delivery of nucleic acid-based therapeutics, J Pharm Sci. (2011 January; 100(1): 38-52. For purposes of the present invention, ASOs are administered intravenously, although other routes of administration are possible, including oral administration. Such alternative routes of administration are applicable to the ASOs described herein.

[0018] Thus, a further aspect of the present invention is pharmaceutical compositions used to administer ASO-T-JAK2 compounds to patients to be treated. These include sterile parenteral dosage forms of the type conventionally used for intravenous administration of drugs. Such pharmaceutical compositions contain excipients that may be required. They may contain one or more diluents, carriers, adjuvants, or combinations thereof, as known in the art for the manufacture of finished dosage forms.

[0019] In one embodiment, the invention provides a method of treating a patient diagnosed with Charcot-Marie-Tooth disease type 2 (CMT2), comprising administering to the patient an ASO-T-IGHMBP2 compound in an amount effective to treat such disease.

[0020] In another embodiment, the invention provides a method for inhibiting expression of a mutated IGHMBP2 gene in an individual carrying a C31401A mutation in the IGHMBP2 gene, comprising administering to the individual an ASO-T-IGHMBP2 compound in an amount effective to inhibit expression of the mutated IGHMBP2 gene in the individual.

[0021] In yet another embodiment, the present invention provides an antisense oligonucleotide (ASO) that targets a nucleic acid molecule encoding a mutant IGHMBP2 gene. Such an ASO can be included in a pharmaceutical composition in combination with a pharma- ceutically acceptable diluent, carrier, adjuvant, or combination thereof.

[0022] Numerous methods exist in the art for preparing an ASO once its nucleotide sequence has been determined. Similarly, to identify the genetic sequence of a gene for which the ASO inhibits protein expression by RNA binding, determination of the appropriate sequence for the ASO-T-IGHMBP2 compound is accomplished by strategies known in the art.

[0023] For the treatment of an individual patient with administration of ASO-TIGHMBP2, the dosage and dosing schedule can be readily determined. Numerous examples of approved administrations of ASOs are known in the art, such as nusinersen (sold as Spinraza), mipomersen (sold as Kynamro), and fomivirsen (sold as Vitravene). In certain circumstances, it may be advantageous to administer ASO-TIGHMBP2 in a derivatized or modified form to optimize bioavailability, duration of action, or other therapeutic effect. Methods for such modification / derivatization are known in the art. See generally, Drug Delivery Trends in Clinical Trails and Translational Medicine: challenges and opportunities in delivery of nucleic acid-based therapeutics, J Pharm Sci. (2011 January; 100(1): 38-52. For purposes of the present invention, ASOs are administered intravenously, although other routes of administration are possible, including oral administration. Such alternative routes of administration are applicable to the ASOs described herein.

[0024] Therefore, a further aspect of the present invention is pharmaceutical compositions used to administer ASO-TIGHMBP2 compounds to patients to be treated. These include sterile parenteral dosage forms of the type conventionally used for intravenous administration of drugs. Such pharmaceutical compositions contain excipients that may be required. They may contain one or more diluents, carriers, adjuvants, or combinations thereof, as known in the art for the manufacture of finished dosage forms.

[0025] These and other features of the present invention will be more readily understood from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings which illustrate various embodiments of the invention. [Brief description of the drawings]

[0026] [Figure 1]Figure 1 shows the results of a bioluminescence assay in which ASO treatment inhibits JAK2 expression and reduces cell viability. [Figure 2A] Figure 2A shows a fluorescent image of SET-2 cells treated with an ASO according to an embodiment of the invention. [Figure 2B] Figure 2B shows a bright field image of SET-2 cells treated with an ASO according to an embodiment of the invention. [Figure 3A] Figure 3A shows a fluorescent image of HEL cells treated with an ASO according to an embodiment of the invention. [Figure 3B] Figure 3B shows a bright field image of HEL cells treated with an ASO according to an embodiment of the invention. [Figure 4A] FIG. 4A shows a fluorescent image of SET-2 cells treated with an ASO according to an embodiment of the invention. [Figure 4B] Figure 4B shows a bright field image of SET-2 cells treated with an ASO according to an embodiment of the invention. [Figure 5A] Figure 5A shows a fluorescent image of HEL cells treated with an ASO according to an embodiment of the invention. [Figure 5B] Figure 5B shows a bright field image of HEL cells treated with ASO according to an embodiment of the present invention. Please note that the drawings of the present invention are not to scale. The drawings are intended to depict only typical aspects of the present invention and therefore should not be considered as limiting the scope of the present invention. In the drawings, like numbers represent like elements between the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention provides target sequences in the JAK2 pre-mRNA that are amenable to binding by synthetic ASOs. Such binding inhibits expression of the JAK2 gene. In patients diagnosed with MDS and PV, such inhibition of JAK2 expression provides an effective method of treating the disorders.

[0028] As mentioned above, both MDS and PV 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 amino acid at position 617 is a valine.

[0029] In one embodiment of the invention, a target region within the JAK2 pre-mRNA molecule is identified that, when bound by an ASO, can inhibit synthesis of the JAK2 protein. This region is comprised of the 19 bp sequence

[0030] [ka]

[0031] and a shorter 16 bp sequence

[0032] [ka]

[0033] Includes.

[0034] The complementary ASO sequences are

[0035] [ka]

[0036] and

[0037] [ka]

[0038] It is.

[0039] These sequences bridge an intron and an exon, respectively, of the JAK2 pre-mRNA, and binding of ASOs containing these sequences to the JAK2 pre-mRNA prevents processing of the pre-mRNA into mature mRNA, which in turn prevents synthesis of the JAK2 protein.

[0040] To test the efficacy of ASOs having SEQ ID NO:4 and / or SEQ ID NO:5 to bind to JAK2 pre-mRNA and inhibit JAK2 protein synthesis, three human cell lines (SET-2, HEL, CMK) are incubated with each of these ASOs and treated with cycloheximide. After 48 hours of incubation, the treated cell lines are harvested and analyzed by CellTiter-Glo. (R) The cells were subjected to (CTG) analysis and RNA extraction. Data regarding the cell lines and culture media are shown in Table 1 below.

[0041] [Table 1]

[0042] Cells are harvested during the growth period and counted using a Cellometer K2. One million cells are incubated in each well of a 6-well plate in the medium listed above and incubated in a humidified incubator at 37° C. with 5% CO2.

[0043] After 24 hours, the medium is changed, ASO is added, and incubation is continued under the same conditions for another 24 hours. Cycloheximide is then added to a final concentration of 0.1 mg / mL. 24 hours after addition of cycloheximide, the medium and cells are combined in a 15 mL conical tube, and a 50 μL aliquot of cells is added to a 96-well assay plate for the CTG assay. Luminescence is recorded using a BioTek Synergy neo2 Multi-mode Reader. The remaining cells are centrifuged at 4°C to collect cell pellets for RNA extraction with Trizol reagent.

[0044] Figure 1 shows the results of the above CTG assay for the SET-2 cell line. Column 1 shows the results for the assay vehicle without ASO. The average relative luminescence units (RLU) are over 3,000,000. RLU is proportional to the number of viable cells in the assay well.

[0045] Column 2 shows the results of the assay when an off-target control ASO (i.e., an ASO that does not target JAK2 pre-mRNA or mRNA) is used. The average RLU is reduced compared to vehicle, but still exceeds 2.5 million.

[0046] Column 3 of Figure 1 shows the results of the assay upon addition of ASOs containing SEQ ID NO:4 or SEQ ID NO:5, where the average RLU is reduced to less than 2,000,000, with some samples dropping to around 1,500,000. This constitutes a significant reduction in RLU and therefore cell viability compared to vehicle.

[0047] These results are confirmed by RNA sequencing using next generation sequencing (NGS) to quantify the expression of the JAK2 gene. Figures 2A and 2B show fluorescent (GFP) and brightfield images, respectively, of assay wells of SET-2 cells treated with ASOs containing SEQ ID NO:4 or SEQ ID NO:5. Figures 3A and 3B show fluorescent and brightfield images, respectively, of HEL cells treated with the same ASOs. In each case, the fluorescence is lower than in untreated cells, demonstrating a reduction in mature JAK2 mRNA in samples incubated with ASOs containing SEQ ID NO:4 or SEQ ID NO:5.

[0048] Accordingly, embodiments of the invention include administering to an individual an ASO comprising SEQ ID NO:4, SEQ ID NO:5, or both, to inhibit expression of JAK2; such administration as a treatment for MDS, PV, or both; ASOs targeting SEQ ID NO:2, SEQ ID NO:3, or both; ASOs having a nucleotide sequence comprising SEQ ID NO:4 or SEQ ID NO:5; and compositions comprising such ASOs in combination with a pharma- ceutically acceptable diluent, carrier, adjuvant, or combination thereof.

[0049] In another aspect, the invention provides target sequences in mutant IGHMBP2 pre-mRNA that are amenable to binding by synthetic ASOs. Such binding inhibits expression of the mutant IGHMBP2 gene. In patients diagnosed with CMT2S who are heterozygous for the mutation, such inhibition may result in "rescue" or return of the IGHMBP2 protein to normal function.

[0050] The complete sequence of the wild type IGHMBP2 gene is shown in the attached sequence listing as SEQ ID NO: 6. The nucleotide at position 31401 is a cytosine. In CMT2S-associated mutations, this nucleotide is an adenine.

[0051] In one embodiment of the invention, a target region within the resulting mutant IGHMBP2 pre-mRNA molecule is identified that, when bound by an ASO, is capable of inhibiting the synthesis of mutant IGHMBP2 protein. The region within the mutant IGHMBP2 gene is a 19 bp sequence that contains the C31401A mutation.

[0052] [ka]

[0053] Includes. The complementary ASO sequence is

[0054] [ka]

[0055] It is.

[0056] An ASO containing sequence ID No. 3 cross-links introns and exons of the mutant IGHMBP2 pre-mRNA and, upon binding to the pre-mRNA, prevents synthesis of the mutant IGHMBP2 protein.

[0057] To test the efficacy of ASOs containing SEQ ID NO:8 in binding to mutant IGHMBP2 pre-mRNA and inhibiting mutant IGHMBP2 protein synthesis, fibroblast cell lines from patients with CMT2S are incubated with ASOs containing SEQ ID NO:8 in RPMI1640 medium supplemented with 10% FBS and treated with cycloheximide. After 48 hours of incubation, treated cells are harvested and subjected to RNA extraction and Western blot analysis to determine IGHMBP2 protein expression.

[0058] More specifically, 1 million cells are incubated in 2 mL of RPMI1640 medium as described above in a humidified incubator at 37° C. with 5% carbon dioxide. After 24 hours, the medium is replaced, ASO is added, and incubation is continued for another 24 hours. Cycloheximide is then added to a final concentration of 0.1 mg / mL. 24 hours after cycloheximide addition, the medium and cells are combined in a 15 mL conical tube, spun, and the cell pellet is collected for RNA extraction with Trizol reagent. RNA extraction is performed, for example, using Qiagen's RNeasy Mini Kit. Western blot analysis is then performed to determine the concentrations of wild-type and mutant proteins in the samples.

[0059] Figures 4A and 4B show fluorescent and bright field micrographs, respectively, of SET-2 cells following treatment with ASOs containing sequence number 8, demonstrating the ability of these ASOs to penetrate the cell membrane.

[0060] Figures 5A and 5B show similar fluorescent and bright field micrographs, respectively, of HEL cells following treatment with an ASO containing SEQ ID NO: 8. These results also demonstrate the ability of such ASOs to penetrate the cell membrane.

[0061] ASOs other than or in addition to ASOs comprising SEQ ID NO:8 may also be useful in practicing the invention. Applicants have identified a "core" 11 nucleotide sequence within SEQ ID NO:8 that provides the basis for sequences with sufficient specificity to allow for identification of binding of ASOs to mutant IGHMBP2 pre-mRNA. As one of skill in the art will appreciate, efficient and effective ASO therapy, such as that described herein, requires the use of ASOs with a length sufficient to reliably identify binding of the ASO to the target sequence. ASOs that include sequences that are 13-35 nucleotides in length and include the core sequence of SEQ ID NO:9 are of sufficient length for use in practicing embodiments of the invention.

[0062] [ka]

[0063] Thus, ASOs having a nucleotide sequence containing 2-24 contiguous nucleotides, including SEQ ID NO:4, are useful in practicing embodiments of the invention, including, for example, SEQ ID NOs:10, 11, and sequences as short as the 13 nucleotide sequence of SEQ ID NO:12.

[0064] [ka]

[0065] Such nucleotide sequences include, for example, the sequences of SEQ ID NO:13 and SEQ ID NO:14, which are 35 nucleotide sequences in length.

[0066] [ka]

[0067] More commonly, ASOs having nucleotide sequences between 13 and 35 nucleotides in length include the core sequence of SEQ ID NO:9 and have as many as 15 upstream nucleotides and / or as many as 12 downstream nucleotides. The ASO "sequence window" is shown in SEQ ID NO:15.

[0068] [ka]

[0069] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0070] This written description uses examples to disclose the invention, including the best mode, and also enables 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 that do not differ insubstantially from the literal language of the claims.

Claims

1. A pharmaceutical composition comprising an ASO-T-JAK2 compound for use in the treatment of myelodysplastic syndromes (MDS), said treatment comprising administering to a patient an effective amount of said ASO-T-JAK2 compound to treat MDS.

2. The pharmaceutical composition according to claim 1, wherein the ASO-T-JAK2 compound targets SEQ ID NO: 2, SEQ ID NO: 3, or both.

3. The pharmaceutical composition according to claim 2, wherein the ASO-T-JAK2 compound comprises a nucleotide sequence comprising SEQ ID NO: 4 or SEQ ID NO:

5.

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

5. A pharmaceutical composition comprising an ASO-T-JAK2 compound for use in the treatment of polycythemia vera (PV), said treatment comprising administering to a patient an effective amount of said ASO-T-JAK2 compound to treat PV.

6. The pharmaceutical composition according to claim 5, wherein the ASO-T-JAK2 compound targets SEQ ID NO: 2, SEQ ID NO: 3, or both.

7. The pharmaceutical composition according to claim 6, wherein the ASO-T-JAK2 compound comprises a nucleotide sequence comprising SEQ ID NO: 4 or SEQ ID NO:

5.

8. The pharmaceutical composition according to claim 5, wherein the amount of the ASO-T-JAK2 compound is an amount sufficient to inhibit the expression of JAK2 in the patient.

9. A pharmaceutical composition comprising an ASO-T-JAK2 compound for use in inhibiting the expression of the JAK2 gene in the treatment of an individual, said treatment comprising administering to the individual an effective amount of said ASO-T-JAK2 compound to inhibit JAK2 expression in the individual, wherein said ASO-T-JAK2 compound targets SEQ ID NO: 2, SEQ ID NO: 3, or both. Claim 10 The pharmaceutical composition according to claim 9, wherein said ASO-T-JAK2 compound comprises a nucleotide sequence comprising SEQ ID NO: 4 or SEQ ID NO:

5. Claim 11 An antisense oligonucleotide (ASO) targeting a nucleic acid molecule encoding JAK2, said ASO targeting SEQ ID NO: 2, SEQ ID NO: 3, or both. Claim 12 The ASO according to claim 11, wherein said ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:

5. Claim 13 The ASO according to claim 11; and A pharmaceutically acceptable diluent, carrier, adjuvant, or combination thereof A pharmaceutical composition comprising the same. Claim 14 The pharmaceutical composition according to claim 13, further comprising a sterile parenteral dosage form.

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