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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/34—Allele or polymorphism specific uses

Definitions

• the present invention relates to certain novel antisense oligonucleotides (ASOs) and, more particularly, to pharmaceutical compositions thereof and to methods for treating certain diseases or conditions in which regulating protein synthesis using ASOs can have therapeutic value. More particularly aspects of the present invention relate to the treatment of known disorders such as polycythemia vera (PV) and myelodysplastic syndrome (MDS), as well as Charcot-Marie-Tooth type 2 (CMT2) disease.
• PV polycythemia vera
• MDS myelodysplastic syndrome
• CMT2 Charcot-Marie-Tooth type 2
• PV is a myeloproliferative disorder characterized by erythroid hyperplasia, myeloid leukocytosis, thrombocytosis, and splenomegaly. Most individuals diagnosed with PV carry a valine-to-phenylalanine mutation at position 617 (V617F) of the JAK2 gene.
• V617F valine-to-phenylalanine mutation at position 617 (V617F) of the JAK2 gene.
• MDS is a group of clonal hematologic stem cell disorders characterized by ineffective hematopoiesis, often resulting in anemia. Some cases of MDS have been associated with the same V617F mutation of the JAK2 gene and specifically with an increased platelet count.
• 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.
• CMT Charcot-Marie-Tooth disease
• CMT also known as hereditary motor and sensory neuropathy
• Symptoms are progressive, often beginning in the feet and lower legs and then the fingers, hands, and arms.
• CMT type 2 (CMT2) is a less common subtype of CMT affecting nerve axons.
• CMT2 CMT type 2S
• IGHMBP2 immunoglobulin mu DNA binding protein 2
• an “antisense oligonucleotide” or “ASO” refers to 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.
• the invention provides a method of treating a patient diagnosed with MDS which comprises administering to the patient an amount of an ASO-T-JAK2 compound effective to treat MDS.
• an ASO-T-JAK2 compound refers to any antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding JAK2.
• ASO antisense oligonucleotide
• an amount of the ASO-T-JAK2 compound effective to treat MDS requires administration of the compound in a quantity effective to inhibit JAK2 protein expression on the patient.
• ASO-T-JAK2 refers to an ASO targeted to a nucleic acid molecule encoding the gene JAK2.
• nucleic acid molecule is a reference to a polynucleotide compound, i.e., RNA.
• An “antisense oligonucleotide” or “ASO” references a synthetic, multi-nucleotide DNA compound that hybridizes to a target RNA in a manner such that gene expression can be inhibited, including by inactivating an mRNA compound.
• Illustrative ASO-T-JAK2 compounds are disclosed herein (SEQ. ID. 4 and 5 targeting SEQ. ID. 2 and 3, respectively).
• 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.
• ASO-T-IGHMBP2 refers to an ASO targeted to a nucleic acid molecule encoding the IGHMBP2 gene.
• nucleic acid molecule is a reference to a polynucleotide compound, i.e., RNA.
• Illustrative 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 a target RNA sequence.
• 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 PV or MDS or CMT2S.
• the invention provides a method of treating a patient diagnosed with PV which comprises administering to the patient an amount of an ASO-T-JAK2 compound effective to treat PV.
• An amount effective of the ASO-T- JAK2 compound effective to treat PV is an amount effective to inhibit JAK2 protein expression in the patient.
• 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 accomplish such inhibition.
• the invention provides novel ASO-T-JAK2 compounds.
• such compounds included, but are not limited to the oligonucleotides of SEQ ID 4 and SEQ ID 5.
• 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.
• 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).
• nusinersen marketed as Spinraza
• mipomersen marketed as Kynamro
• fomivirsen marketed as Vitravene
• it may be advantageous to administer the ASO- T-JAK2 in a derivatized or modified form in order to optimize bioavailability, 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.
• the ASOs are administered intravenously, though other routes of administration are possible, including oral administration. Such alternative routes of administration are
• 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.
• the invention provides a method of treating a patient diagnosed with Charcot-Marie -Tooth type 2 (CMT2) which comprises: administering to said patient an amount of an ASO-T-IGHMBP2 compound effective to treat such disease.
• CMT2 Charcot-Marie -Tooth type 2
• the invention provides a method of inhibiting expression of a mutated IGHMBP2 gene in an individual carrying a C31401 A mutation of the IGHMBP2 gene, the method comprising: administering to the individual an amount of an ASO-T-IGHMBP2 compound effective to inhibit expression of the mutant IGHMBP2 gene in the individual.
• the invention provides an antisense oligonucleotide (ASO) targeted to a nucleic acid molecule encoding a mutated IGHMBP2 gene.
• ASO antisense oligonucleotide
• Such an ASO may be included in a pharmaceutical composition in combination with a pharmaceutically acceptable diluent, carrier, adjuvant, or combination thereof.
• ASO-T- IGHMBP2 In the treatment of an individual patient by administration of an ASO-T- IGHMBP2, 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).
• nusinersen marketed as Spinraza
• mipomersen marketed as Kynamro
• fomivirsen marketed as Vitravene
• compositions that are used for the administration of the ASO-T- IGHMBP2 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.
• FIG. 1 shows the results of a bioluminescence assay in which ASO treatment inhibits JAK2 expression and decreases cell viability
• FIGS. 2A and 2B show, respectively, fluorescent and brightfield images of SET-2 cells treated with ASOs according to embodiments of the invention
• FIGS. 3A and 3B show, respectively, fluorescent and brightfield images of HEL cells treated with ASOs according to embodiments of the invention
• FIGS. 4A and 4B show, respectively, fluorescent and brightfield images of SET-2 cells treated with ASOs according to embodiments of the invention.
• FIGS. 5A and 5B show, respectively, fluorescent and brightfield images of HEL cells treated with ASOs according to embodiments of the invention.
• the present invention provides 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 MDS and PV, such inhibition of JAK2 expression provides an effective method of treatment of the disorder.
• MDS and PV 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.
• 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 a 19 bp sequence:
• TTT CCTT AGT CTTT CTTT G (SEQ ID NO 2) and the shorter 16 bp sequence:
• Each of these sequences bridges an intron and exon of the JAK2 pre-mRNA.
• ASOs containing these sequences are bound to JAK2 pre-mRNA, processing of the pre-mRNA into mature mRNA is prevented, as is synthesis of the JAK2 protein.
• Cells are harvested during the growth period and counted using a Cellometer K2. One million cells are incubated in the listed medium in each well of a six-well plate and incubated in a humidified incubator at 37 °C with 5% C0 2 .
• the medium is changed, the 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. Twenty-four hours after addition of the cycloheximide, the medium and cells are combined in a 15 mL conical tube and 50 pL aliquots of cells are added to a 96-well assay plate for CTG assaying. Luminescence is recorded using a BioTek Synergy neo2 Multi-mode Reader. The remaining cells are spun at 4 °C to collect a cell pellet for RNA extraction by Tryzol reagent.
• FIG. 1 shows results of the CTG assay described above for the SET-2 cell line.
• the first column is the result for the assay vehicle, which contains no ASOs.
• Average relative luminescence units (RLUs) are in excess of 3,000,000. RLU is proportional to the number of viable cells in the assay well.
• the second column shows the results of the assay when off-target control ASOs (i.e., ASOs not targeting JAK2 pre-mRNA or mRNA) are used. Average RLUs are reduced as compared to vehicle, but are still in excess of 2,500,000.
• off-target control ASOs i.e., ASOs not targeting JAK2 pre-mRNA or mRNA
• the third column of FIG. 1 shows the results of the assay when ASOs containing SEQ ID NO 4 or SEQ ID NO 5 are added.
• average RLUs are reduced to less than 2,000,000, with some samples exhibiting a reduction to approximately 1,500,000. This constitutes a significant reduction in RLUs, and consequently cell viability, as compared to vehicle.
• FIGS. 2A and 2B show, respectively, fluorescent (GFP) and brightfield images of assay wells for SET -2 cells treated with ASOs containing SEQ ID NO 4 or SEQ ID NO 5.
• FIGS. 3 A and 3B show, respectively, fluorescent and brightfield images for HEL cells treated with the same ASOs. In each case, fluorescence is lower than in untreated cells, demonstrating decreased mature JAK2 mRNA in samples incubated with ASOs containing SEQ ID NO 4 or SEQ ID NO 5.
• embodiments of the invention include the administration of an ASO containing SEQ ID NO 4, SEQ ID NO 5, or both, to an individual in order 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 nucleotide sequences that include SEQ ID NO 4 or SEQ ID NO 5; and compositions containing such ASOs in combination with pharmaceutically acceptable diluents, carriers, adjuvants, or combinations thereof.
• the present invention provides target sequences in the mutant IGHMBP2 pre-mRNA that are amenable to binding by a synthetic ASO. When so bound, expression of the mutant IGHMBP2 gene is inhibited. In patients diagnosed with CMT2S who are heterozygous for the mutation, such inhibition may result in the “rescue” or return to normal function of the IGHMBP2 protein.
• the full sequence of the wildtype IGHMBP2 gene is shown in the attached sequence listing as SEQ ID NO 6.
• the nucleotide at position 31401 is cytosine. In the mutation associated with CMT2S, this nucleotide is adenine.
• a target region within the resulting mutant IGHMBP2 pre-mRNA molecule is identified that, when bound with an ASO, is capable of inhibiting synthesis of the mutant IGHMBP2 protein.
• That region within the mutant IGHMBP2 gene comprises a 19-bp sequence that includes the C31401 A mutation:
• An ASO comprising SEQ ID NO 3 bridges an intron and exon of the mutant IGHMBP2 pre-mRNA and, when bound to the pre-mRNA, prevents synthesis of the mutant IGHMBP2 protein.
• a fibroblast cell line of a patient with CMT2S is incubated with an ASO including SEQ ID NO 8 in RPMI 1640 medium supplemented with 10% FBS and treated with cycloheximide. After a 48-hour incubation, the treated cells are collected and for RNA extraction and Western Blot analysis to determine IGHMBP2 protein expression.
• RNA extraction is carried out using, for example, Qiagen’s RNeasy Mini Kit. Western Blot analysis is then carried out to determine the concentration of wild type and mutant proteins in the samples.
• FIGS. 4A and 4B show fluorescent and brightfield photomicrographs, respectively, of SET-2 cells following treatment with ASOs including SEQ ID NO 8, demonstrating the ability of these ASOs to penetrate the cell membrane.
• FIGS. 5A and 5B show similar fluorescent and brightfield photomicrographs, respectively, of HEL cells following treatment with ASOs including SEQ ID NO 8. Again, these results demonstrate the ability of such ASOs to penetrate the cell membrane.
• ASOs other than or in addition to those including SEQ ID NO 8 may be useful in practicing the invention.
• Applicant has identified a “core” 11 nucleotide sequence within SEQ ID NO 8 that would provide a basis for sequences having sufficient specificity to enable discriminate binding of an ASO to the mutant IGHMBP2 pre- mRNA.
• efficient and effective ASO therapies such as those described herein require the use of ASOs having a length sufficient to ensure discriminate binding of the ASO to the target sequence.
• ASOs including sequences of between 13 nucleotides and 35 nucleotides in length and including the core sequence of SEQ ID NO 9 are of sufficient length for use in practicing embodiments of the invention.
• nucleotide sequences that includes SEQ ID NO 4 and between two and 24 adjacent nucleotides are useful in practicing embodiments of the invention.
• nucleotide sequences include, for example, sequences as short as the 13-nucleotide sequences of SEQ ID NOS, 10, 11, and 12:
• nucleotide sequences also include, for example, sequences as long as the 35- nucleotide sequences of SEQ ID NOS 13 and 14.
• ASOs having a nucleotide sequence between 13 nucleotides and 35 nucleotides in length comprising the core sequence of SEQ ID NO 9 and as many as 15 upstream nucleotides and/or 12 downstream nucleotides.
• the ASO “sequence window” is shown in SEQ ID NO 15.

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