Antisense therapy for ptp1b-related disorders

Abstract

An isolated or purified antisense oligomer targeted to a nucleic acid molecule encoding a PTPN1 pre-mRNA, wherein the antisense oligomer inhibits expression of PTP1B.

Description

Technical Field

[0001] This invention relates to antisense oligomers (ASOs) that promote the modification of isoforms of the protein tyrosine phosphatase-1B (PTP1B) encoded by the gene PTPN1. The invention further provides a method for treating, preventing, or improving the effects of insulin resistance and leptin resistance by administering the antisense oligomer (ASO) and a therapeutic composition containing the antisense oligomer to the PTPN1 gene. Background Technology

[0002] The following discussion of the background art is intended only to facilitate understanding of the invention. This discussion is not an admission or endorsement that any material mentioned was or was part of common general knowledge at the priority date of the application.

[0003] Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by insufficient insulin secretion or inefficient insulin processing. Insulin, a hormone secreted by pancreatic β-cells, plays a central role in glucose metabolism. Insulin deficiency leads to elevated plasma glucose concentrations, and a persistent hyperglycemic state results in hyperglycemia, ultimately progressing to T2DM. Insulin deficiency stems from upstream and downstream dysregulations, termed pancreatic β-cell dysfunction (upstream) and insulin resistance (downstream), respectively. β-cell dysfunction leads to reduced insulin production, while insulin resistance is described as a disruption occurring during insulin signaling pathways in glucose-sensing cells. Upstream and downstream insulin dysregulations interact with each other in a complex relationship and jointly contribute to the pathogenesis of T2DM. In summary, β-cell dysfunction and insulin resistance are the two fundamental causes of T2DM.

[0004] Obesity is a key accelerating factor in type 2 diabetes mellitus (T2DM) and is characterized by leptin resistance. Leptin is an adipocyte-derived hormone that acts on the hypothalamus to reduce food intake and increase energy expenditure. Obesity is associated with a disruption (leptin resistance) occurring during the leptin signaling pathway, which leads to increased food intake and decreased energy expenditure.

[0005] PTP1B expression negatively regulates both the insulin signaling pathway and the leptin signaling pathway, therefore PTP1B is a therapeutic target for type 2 diabetes mellitus (T2DM) and obesity.

[0006] Conventional treatment for type 2 diabetes mellitus (T2DM) is based on a variety of oral hypoglycemic agents, which are currently designated antidiabetic drug therapies and are generally designed to lower blood glucose levels by targeting one or more of six key organs and / or tissues (i.e., pancreas, liver, skeletal muscle, small intestine, kidneys, and adipose tissue). To date, no single drug has shown promise in addressing the underlying causes of T2DM (i.e., pancreatic β-cell dysfunction and insulin resistance).

[0007] PTP1B functions as an oncogene. The PTP1B gene is commonly amplified in ovarian, gastric, prostate, and breast cancers and is associated with poor prognosis. Knockdown of PTP1B reduces cell growth, induces cell cycle arrest and apoptosis, and reduces cancer cell migration and invasion by reversing the epithelial-mesenchymal transition (EMT) process. Therefore, PTP1B is also a therapeutic target for solid tumors.

[0008] The present invention seeks to provide compositions and methods for reducing the effects of insulin resistance, type 2 diabetes mellitus (T2DM), leptin resistance, obesity, and solid tumors, or to provide consumers with useful or commercial options. Invention Overview

[0010] This invention is based on a surprising discovery: the use of isolated or purified antisense oligomers (ASO) can lead to a reduction in the production of functional PTP1B protein. These antisense oligomers are used to modify the splicing production of PTPN1 precursor mRNA to increase the production of truncated, nonsense, or prematurely terminated proteins (such as proteins with premature stop codons).

[0011] In summary, according to one aspect of the invention, an isolated or purified antisense oligomer (ASO) is provided for modifying the splicing of precursor mRNA in the protein tyrosine phosphatase-1B (PTP1B) encoded by the PTPN1 gene transcript or a portion thereof. Preferably, an isolated or purified antisense oligomer is provided for inducing splicing regulation, particularly causing exon skipping of premature stop codons, thereby resulting in a reduction in the production of the full-length PTPN1 gene transcript or a portion thereof.

[0012] Preferably, the antisense oligomer is a phosphatidylmorpholino oligomer.

[0013] Preferably, the antisense oligomer is selected from the group comprising the sequences listed in Table 1. Preferably, the antisense oligomer is selected from the list comprising: SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41. Preferably, the antisense oligomer used in the present invention is selected from the list comprising: SEQ ID NO: 1, or 32-36. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 33.

[0014] According to a further aspect of the invention, the invention extends to cDNA or clonal copies of the antisense oligomer sequence of the invention, and vectors containing the antisense oligomer sequence of the invention. The invention also extends further to cells containing such sequences and / or vectors.

[0015] A method for manipulating splicing factor binding in PTPN1 gene transcripts is also provided, the method comprising the following steps:

[0016] Provide one or more of the antisense oligomers as described herein, and allow the oligomers to bind to the target nucleic acid site.

[0017] Also provided is a medicament, preventive or therapeutic composition for treating, preventing or improving the effects of a disease associated with the PTP1B protein in a subject, the composition comprising:

[0018] One or more antisense oligomers as described herein and

[0019] One or more drug-acceptable carriers and / or diluents.

[0020] Preferably, the disease conditions associated with PTP1B protein are insulin resistance, type 2 diabetes (T2DM), leptin resistance, obesity, and solid tumors.

[0021] Subjects with diseases associated with the PTP1B protein can be mammals, including humans.

[0022] A method for treating, preventing, or improving the effects of diseases associated with the PTP1B protein is also provided, the method comprising the following steps:

[0023] An effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein is administered to a subject.

[0024] The use of purified and isolated antisense oligomers, as described herein, for the preparation of drugs for the treatment, prevention, or improvement of the effects of diseases associated with the PTP1B protein is also provided.

[0025] Kits for treating, preventing, or improving the effects of diseases associated with the PTP1B protein in subjects are also provided, which contain at least an antisense oligomer as described herein, and combinations or mixtures thereof packaged in a suitable container, along with instructions for use.

[0026] Further aspects of the invention will now be described with reference to the accompanying non-limiting examples and drawings.

[0027] Brief description of the attached diagram

[0028] Further features of the invention are described more fully in the following description of several non-limiting embodiments of the invention. This description is given for illustrative purposes only and should not be construed as limiting the broad overview, disclosure, or description of the invention as described above. The description will be made with reference to the accompanying drawings, in which:

[0029] Figure 1 These are exon maps of human PTPN1-201 transcripts and mouse Ptpn1-201 transcripts.

[0030] Figure 2 This is a Northern blot showing the transfection efficiency of AO 1-8 at a concentration of 400 nanomolars in the Huh-7 cell line. S: scrambled sequence, UT: untreated, NC: negative control, Imax: RNAiMAX, pro: PRO, si: SI + .

[0031] Figure 3 This is a Northern blot showing the transfection efficiency of AO 9-16 at a concentration of 400 nanomolar in the Huh-7 cell line using RNAiMAX reagent. S: scrambled sequence, UT: untreated, NC: negative control.

[0032] Figure 4 This is a Northern blot showing the transfection efficiency of AO 17-30 at a concentration of 400 nanomolar in the Huh-7 cell line using L3K reagent. S: scrambled sequence, UT: untreated, NC: negative control.

[0033] Figure 5 This shows the Northern blots of the dose-response assays for AO1 and AO4. Concentrations in the Huh-7 cell line included 400, 200, 100, 50, 25, and 12.5 nanomolars. S: scrambled sequence, UT: untreated, NC: negative control.

[0034] Figure 6 This is a schematic diagram of a modified transfection protocol for HepG2 transfection experiments based on the manufacturer's instructions for RNAiMAX and Lipofectamine 3000 (L3K). AO1 in 2' OMePS form is used in this experiment. The stock concentration of AO1 is 181171 nanomolars. Opti is Opti-MEM. TM I is an abbreviation for reduced serum culture medium.

[0035] Figure 7 This is a Northern blot showing the experiments conducted in the HepG2 cell line with different transfection reagents (RNAiMAX and L3K) and different transfection protocols (RNAiMAX: 1.1-1.7; L3K: 2.1 and 2.2), and the results indicate that protocol 1.3 (the reverse transfection protocol of RNAiMAX) is the optimal protocol for transfecting 2'OMePS antisense oligonucleotides into HepG2. The 2'OMePS form of AO1 was used in this experiment.

[0036] Figure 8 This is a Northern blot showing the comparison of exon 2-hopping efficiency between PTPN1 1E2A(+1+25)(AO1), the 2'-OMePS form of ISIS107773, PTPN1 1E2A(+1+23)(AO 31), and PTPN1 1E2A(+3+27)(AO 32) in HepG2 at a concentration of 400 nanomolar.

[0037] Figure 9 This is a representation of the Sanger sequencing results, which confirm that AO1, PTPN1 1E2A(+1+25) induce exon 2 skipping during the transcription of the PTPN1 gene.

[0038] Figure 10 This is a Northern blot showing the comparison of exon 2-jumping efficiency and non-jumping product knockdown efficiency between the 2'-OMePS forms of PTPN1 1E2A(+1+25)(AO1), PTPN1 1E2A(+3+27)(AO 32), and ISIS 107773 (5-10-5MOE gapmer) in HepG2, in triplicate at a concentration of 400 nanomolar.

[0039] Figure 11 This shows the Northern blot of the dose-response assay for PTPN1 1E2A(+1+25)(AO 1). In HepG2, the concentrations included 400, 200, 100, 50, 25, 12.5, 6.3, and 3.1 nanomolars.

[0040] Figure 12 This is a Northern blot showing the transfection efficiency of AO 1, 32-36 at a concentration of 400 nanomolar in the IHH cell line using RNAiMAX reagent.

[0041] Figure 13 This is a Northern blot comparing the exon 2 skipping efficiency and non-skipping product knockdown efficiency of AO 1, AO 32-36, and ISIS 107773 in the 2'-OMePS form with ISIS 107773 (5-10-5 MOE gapmer) at a concentration of 400 nanomolar using RNAiMAX reagent in the hepG2, Huh-7, and IHH cell lines.

[0042] Figure 14This is a Northern blot illustrating the dose-response assay of the 2'OMePS form of PTPN1 1E2A(+5+29)(AO 33)(Diabexa-2) in HepG2 and IHH cells. Concentrations included were 400, 200, 100, 50, 25, and 12.5 nanomoles. Cells were transfected using RNAiMAX.

[0043] Figure 15 This is a Northern blot illustrating the dose-response assay of the PMO form of PTPN1 1E2A (+5+29)(AO 33)(Diabexa-2) in IHH cells. Concentrations included 30, 15, and 7.5 μmol. Cells were transfected via nuclear transfection.

[0044] Figure 16 This shows a reduced Western blot of PTP1B protein production induced in IHH cells by 20 OMePS (400 nmol) of AO33 (Diabexa-2) and PMO (15, 7.5 μmol) of AO33 (Diabexa-2). Cells were harvested 72 hours after AO transfection.

[0045] Figure 17 This is a Northern blot showing the transfection efficiency of AO 37-41 (AO targeting mouse Ptpn1 exon 2) at a concentration of 400 nanomolar in the HepG2 cell line using RNAiMAX reagent.

[0046] Figure 18 This is a Northern blot showing the transfection efficiency of AO 37-41 (AO targeting mouse Ptpn1 exon 2) and AO 1, 32, 33 (AO targeting human PTPN1 exon 2) at a concentration of 400 nanomolar AO in mouse AML-12 cell lines using RNAiMAX or L3K reagent.

[0047] Figure 19 This is a Northern blot showing the transfection efficiency of AO 37, 38, 41 (AO targeting mouse Ptpn1 exon 2) and AO 1, 32, 33 (AO targeting human PTPN1 exon 2) in the mouse AML-12 cell line using RNAiMAX. 19A: AO 37 is the mouse version of AO 1 (three mismatches), AO 38 is the mouse version of AO 32 (three mismatches), and AO 41 is the mouse version of AO 33 (two mismatches); 19B: Transfection efficiency of AO 37, 38, 41, 1, 32, 33 at a concentration of 400 nanomolars; 19C: Dose dependence of AO 38; 19D: Dose dependence of AO 41.

[0048] Figure 20 This is an image showing PTPN1 expression in cancer cells. Annealing temperatures for the RT-PCR reactions included 57.8°C, 60°C, and 62°C. The PCR cycle was 30. Invention Details

[0050] Detailed description of the invention

[0051] antisense oligomers

[0052] This invention is based on the surprising discovery that altering the expression of protein tyrosine phosphatase-1B (PTP1B), encoded by the gene PTPN1, can mediate the effects of insulin resistance, type 2 diabetes mellitus (T2DM), leptin resistance, obesity, and solid tumors. This alteration of PTP1B expression can be achieved using antisense oligomers (also known as antisense oligonucleotides, AOS, AO, and AON; these terms are interchangeable).

[0053] Protein tyrosine phosphatase-1B (PTP1B), encoded by the gene PTPN1, is a phosphatase that negatively regulates insulin signaling, thus leading to insulin resistance (one of the root causes of type 2 diabetes mellitus). In addition to blocking insulin signaling, PTP1B also downregulates the leptin signaling pathway, resulting in decreased energy expenditure and increased fat accumulation associated with obesity, which contributes to insulin resistance and is one of the most important risk factors for type 2 diabetes mellitus. Since PTP1B simultaneously blocks both insulin and leptin signaling, this invention investigates the target gene for the development of therapeutic agents for type 2 diabetes mellitus and obesity using PTPN1.

[0054] Without being bound by any theory, this invention is based on the following understanding:

[0055] Downregulation of PTP1B expression leads to upregulation of insulin signaling; and / or

[0056] Downregulation of PTP1B expression leads to upregulation of the leptin signaling pathway.

[0057] PTP1B protein is also associated with a variety of solid tumor cancers, and knocking down PTP1B reduces cell growth, induces cell cycle arrest and apoptosis, and reduces cancer cell migration and invasion by reversing the epithelial-mesenchymal transition (EMT) process.

[0058] PTPN1 has ten exons, including four exons (exons 2, 3, 8, and 9) containing residue overlap splicing sites. Figure 1Two of these exons (exon 2 and exon 3) are located near the 5' end of the transcript. If exon 2 is skipped, a premature stop codon is induced in exon 3, suggesting that the variant transcript produced by exon 2 skipping may not be translated into a functional PTP1B protein. Alternatively, exon skipping can be used to develop truncated or nonsense PTP1B proteins.

[0059] Preferably, the diseases or conditions treated or prevented by the antisense oligomers of the present invention are: (i) diseases associated with downregulation of insulin signaling in the subject; (ii) diseases associated with downregulation of the leptin signaling pathway in the subject; and / or (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the disease may be T2DM, obesity, or cancer.

[0060] This invention does not specifically seek to affect the overall expression of PTP1B protein, for example, by blocking or removing all PTPN1 transcripts. Rather, it seeks to increase the production of truncated, nonsense, or prematurely terminated proteins. The overall production of PTPN1 RNA molecules may not change significantly (although some variations may occur). Preferably, these truncated, nonsense, or prematurely terminated proteins lack one or more functional domains involved in biocatalytic processes. For example, exons 1, 2, 3, 4, 5, and 6 collectively encode a tyrosine protein phosphatase motif, and the translated protein lacking this domain may be unable to catalyze the removal of phosphate groups from phosphorylated tyrosine residues on the protein. Exons 6 and 7 encode regions containing substrate-binding sites, and removal of these exons may result in nonfunctional PTP1B proteins.

[0061] Preferably, the protein contains an internally truncated portion (i.e., a protein lacking the amino acids encoded by one or more exons). If the PTP1B protein is knocked out, there may be a problem with increased PTPN1 transcription as the body attempts to compensate for the reduction in the total amount of PTP1B protein. Conversely, the presence of an internally truncated portion (preferably a protein lacking one or more features of the complete PTP1B protein) should be sufficient to prevent increased transcription, but still provide a therapeutic advantage due to the reduction in the total amount of functional PTP1B protein. Preferably, exon skipping leads to exon 2 skipping; exon 2 skipping leads to the induction of a premature stop codon in exon 3.

[0062] The antisense oligomer-induced exon skipping of the present invention does not require complete or even substantial elimination of the function of the PTP1B protein. Preferably, the exon skipping process results in a reduction or impairment of the function of the PTP1B protein.

[0063] Unlike other antisense oligomer-based therapies, this invention does not induce increased RNA degradation by recruiting RNase H, which preferentially binds to the degraded RNA and binds to the DNA of the PTPN1 gene in a double-stranded form. This invention also does not rely on the hybridization of the antisense oligomer to the PTPN1 genomic DNA or the binding of the antisense oligomer to mRNA to regulate the amount of PTP1B protein produced by interfering with normal functions (such as replication, transcription, translocation, and translation).

[0064] Instead, antisense oligomers are used to modify the transcription process to increase the production of truncated, nonsense, or prematurely terminated proteins. Preferably, the invention causes a skipping of exon 2 to induce a premature stop codon in exon 3. This will result in variant transcripts that may not be translated into the functional PTP1B protein.

[0065] Preferably, the antisense oligomer targets the splicing site in the PTPN1 gene. The target site may also include some flanking sequences surrounding the splicing site.

[0066] Antisense oligomers can also, or alternatively, bind to polyadenylation sites. Target sites can also be located near polyadenylation sites but not overlapping them; that is, they can alternatively cover sequences upstream or downstream of the polyadenylation site, and in these cases, the antisense oligomer may nonspecifically cover the polyadenylation site. Localization to the vicinity of the polyadenylation site is sufficient to disrupt the ability of the cleavage factor to bind to the polyadenylation site.

[0067] According to a first aspect of the invention, an antisense oligomer is provided that is capable of binding to selected targets on the PTPN1 gene transcript to modify the splicing of precursor mRNA in the PTPN1 gene transcript or a portion thereof.

[0068] For example, in one aspect of the invention, an antisense oligomer of 10 to 50 nucleotides is provided, comprising a targeting sequence complementary to a region near or within a splice site and / or polyadenylation site of the PTPN1 precursor mRNA.

[0069] The terms “antisense oligomer” and “antisense compound”, as well as “antisense oligonucleotide” and “ASO”, are used interchangeably and refer to the sequence of cyclic subunits, each subunit carrying a base-pairing portion linked by inter-subunit bonds, thereby allowing the base-pairing portion to hybridize with a target sequence in a nucleic acid (typically RNA) via Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunit is based on ribose or another pentose, or, in a preferred embodiment, on a morpholino group (see description of morpholino oligomers below). The oligomer can have exact or near sequence complementarity with the target sequence; variations in the sequence near the oligomer's end are generally preferred over variations in the internal sequence. The terms “precursor RNA” and “precursor mRNA” are used interchangeably.

[0070] The term "isolated" means material that is substantially or substantially free of the components typically present in its natural state. For example, as used herein, "isolated polynucleotide" or "isolated oligonucleotide" can refer to a purified polynucleotide, or a polynucleotide that has been side-linked from its natural state, such as a DNA fragment removed from a sequence adjacent to a segment in the genome. When the term "isolation" refers to cells, it means the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with a polynucleotide duplication disease). In the context of mRNA or protein, "isolation" means the recovery of mRNA or protein from a source (e.g., cells).

[0071] Antisense oligomers can be considered as “pointing to” or “targeting” the target sequence with which they hybridize. In some embodiments, the target sequence includes a region containing a splice site and / or a polyadenylation site, as well as a surrounding region. Target sequences typically include the AUG start codon of mRNA, a splice site of a translation repressor oligomer or pretreated mRNA, or a region of a splice repressor oligomer (SSO). Target sequences of splice sites can include mRNA sequences having 1 to 25 base pairs at their 5' end, which are downstream of the normal splice acceptor junction in pretreated mRNA. Preferred target sequences are any regions of pretreated mRNA that include a splice site, are entirely contained within an exon coding sequence, or span a splice acceptor or donor site. When an oligomer targets a target nucleic acid in the manner described above, it is more generally considered to “target” a biologically relevant target, such as a protein, virus, or bacteria.

[0072] As used herein, “sufficient length” refers to an antisense oligonucleotide complementary to at least 8 (more commonly 8-30) consecutive nucleotides in the target PTPN1 precursor mRNA. In some embodiments, a sufficient length antisense includes at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides in the target PTPN1 precursor mRNA. In some embodiments, a sufficient length antisense includes at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides in the target PTPN1 precursor mRNA. A sufficiently long antisense oligonucleotide has at least a minimum number of nucleotides to enable specific hybridization with exon 2. Preferably, the oligonucleotide of sufficient length is about 10 to about 50 nucleotides, including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 or more nucleotides. In one embodiment, the oligonucleotide of sufficient length is 10 to about 30 nucleotides. In another embodiment, the oligonucleotide of sufficient length is 15 to about 25 nucleotides. In yet another embodiment, the oligonucleotide of sufficient length is 20 to 30, or 20 to 50 nucleotides. In yet another embodiment, the oligonucleotide of sufficient length is 22 to 28, 25 to 28, 24 to 29, or 25 to 30 nucleotides.

[0073] In some embodiments, the antisense oligomer has sufficient sequence complementarity with the target RNA (i.e., the splicing factor binding site-selected regulated RNA) to effectively block regions of the target RNA (e.g., precursor mRNA). In exemplary embodiments, such blocking of PTPN1 precursor mRNA is used to further regulate splicing by masking the binding site of the native protein and / or to regulate or modify splicing by altering the structure of the target RNA. In some embodiments, the target RNA is a target precursor mRNA (e.g., PTPN1 gene precursor mRNA).

[0074] An antisense oligomer that has sufficient sequence complementarity to the target RNA sequence to regulate the binding of splicing factors to the target RNA means that the antisense oligomer has a sequence sufficient to trigger the masking of the binding site of the natural protein, thereby causing further truncation of the PTP1B protein and / or alteration of the three-dimensional structure of the target RNA.

[0075] The selected antisense oligomer can be made shorter (e.g., about 12 bases) or longer (e.g., about 50 bases) and includes a small number of mismatches, provided that the sequence is sufficiently complementary when hybridizing with the target sequence to affect splicing factor binding regulation, and optionally forms an RNA antisense oligomer heteroduplex with the target sequence at a Tm of 45°C or higher.

[0076] Preferably, the antisense oligomer is selected from the group comprising the sequences listed in Table 1. Preferably, the antisense oligomer is selected from the list comprising: SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41. Preferably, the antisense oligomer used in the present invention is selected from the list comprising: SEQ ID NO: 1, or 32-36. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 33. Preferably, the antisense oligomer causes exon hopping in exon 2.

[0077] In some implementations, the complementarity between the target sequence and the antisense oligomer is sufficient to form a stable double strand. The complementary region between the antisense oligomer and the target RNA sequence can be as short as 8-11 bases, but can also be 12-15 bases or more, for example, 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. Antisense oligomers of approximately 16-17 bases are generally long enough to have a unique complementary sequence. In some implementations, a minimum length of complementary bases may be required to achieve the necessary binding Tm, as discussed herein.

[0078] In some implementations, oligonucleotides up to 50 bases in length may be suitable, wherein at least a minimum number of bases (e.g., 10-12 bases) are complementary to the target sequence. However, generally, when the length of the oligonucleotide is less than about 30 bases, promoted or active uptake in the cell is optimized. For the phosphatidylmorpholino oligomer (PMO) antisense oligomers further described herein, binding stability and uptake are typically optimally balanced when the length is 18-25 bases. This includes antisense oligomers consisting of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases (e.g., PMO, PMO-X, PNA, LNA, 2'-OMe).

[0079] In some implementations, the antisense oligomer can be 100% complementary to the target sequence, or may include mismatches, for example, to accommodate variants, provided that the heteroduplex formed between the antisense oligomer and the target sequence is sufficiently stable to withstand cellular nucleases and other degradation mechanisms that may occur in vivo. Therefore, some oligonucleotides may have about or at least about 70% sequence complementarity with the target sequence, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity.

[0080] Mismatches towards the end regions of the hybrid duplex (if present) are generally less stable than those in the middle regions. According to the well-known principle of duplex stability, the permissible number of mismatches will depend on the length of the antisense oligomer, the percentage of G:C base pairs in the duplex, and the location of the mismatch within the duplex. While such antisense oligomers are not necessarily 100% complementary to the target sequence, stable and specific binding to the target sequence is effective, allowing for the regulation of splicing factors binding to the target precursor mRNA.

[0081] The stability of the duplex formed between the antisense oligomer and the target sequence depends on the binding Tm and the sensitivity of the duplex to cellular enzyme cleavage. The Tm of the oligonucleotide relative to the complementary RNA sequence can be measured using conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or those described in Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154, pp. 94-107. In some embodiments, the binding Tm of the antisense oligomer relative to the complementary RNA sequence can be above body temperature, and preferably above about 45°C or 50°C. Tm in the temperature range of 60-80°C or higher is also included.

[0082] Additional examples of variants include antisense oligomers having about or at least about 70% sequence identity or homology over the entire length of any one of SEQ ID NOs: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or homology.

[0083] More specifically, an antisense oligomer is provided that can bind to a selected target site to regulate or modify splicing in or in part of the PTPN1 gene transcript. The antisense oligomer is preferably selected from those provided in Table 1. Preferably, the antisense oligomer is selected from the list comprising: SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41. Preferably, the antisense oligomer used in this invention is selected from the list comprising: SEQ ID NO: 1, or 32-36. More preferably, the antisense oligomer used in this invention is SEQ ID NO: 33.

[0084] The antisense oligomer-induced splicing factor blocking of the present invention does not require complete or even substantial reduction of the amount of PTP1B produced.

[0085] Table 1. List of SEQ IDs for antisense oligomers targeting human PTPN1 or mouse Ptpn1

[0086]

[0087]

[0088]

[0089] The reverse complementary sequence is shown as 5'-3'. The reference point (0) is set at the first base of the polyadenylation signal; therefore, "+" indicates A. 0 The sequence downstream of ATAAA, with a "-" indicating the upstream sequence.

[0090] Preferably, the antisense oligomer is selected from the list comprising: SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41. More preferably, the antisense oligomer used in the present invention is selected from the list comprising: SEQ ID NO: 1, or 32-36. Most preferably, the antisense oligomer used in the present invention is SEQ ID NO: 33.

[0091] How to use

[0092] The present invention further provides a method for manipulating the binding of splicing factors in the PTPN1 gene transcript, the method comprising the following steps:

[0093] a) Provide one or more of the antisense oligomers as described herein, and allow the oligomers to bind to the target nucleic acid site.

[0094] According to another aspect of the invention, a splicing factor binding modified target nucleic acid sequence of PTPN1 is provided, the sequence comprising a DNA equivalent of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 and a complementary sequence thereof. Preferably, the antisense oligomer causes exon skipping of exon 2.

[0095] Designing antisense oligomers to completely mask splice sites and / or polyadenylation sites may not be necessary for changes in the proportion of truncated, nonsense, or prematurely terminated proteins. Furthermore, when designing antisense oligomers, the inventors have found that the size or length of the antisense oligomer itself is not always a primary factor. For some targets, antisense oligomers as short as 20 bases can induce cleavage modifications, and in some cases, antisense oligomers as short as 20 bases are more effective than other, longer (e.g., 25 bases) oligomers targeting the same region.

[0096] More specifically, the antisense oligomers may be selected from those listed in Table 1. These sequences are preferably selected from any one or more of SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 and combinations thereof. This includes sequences that can hybridize with such sequences under strict hybridization conditions, sequences complementary to such sequences, sequences containing modified bases, sequences with modified backbones, and their functional truncations or extensions that control or regulate RNA processing activity in the PTPN1 gene transcript. Preferably, the ASO used in this invention is selected from the list including: SEQ ID NO: 1, or 32-36. More preferably, the antisense oligomer used in this invention is SEQ ID NO: 33. Preferably, the antisense oligomer causes exon skipping in exon 2.

[0097] Antisense oligomers are complementary to DNA, cDNA, or RNA when a sufficient number of corresponding positions in each molecule are occupied by nucleotides capable of hydrogen bonding with each other. Therefore, "specific hybridization" and "complementarity" are terms used to indicate a sufficient degree of complementarity or pairing that results in stable and specific binding between the oligomer and its DNA, cDNA, or RNA target. Those skilled in the art will understand that the sequence of an antisense oligomer does not need to be 100% complementary to the sequence of its specific hybridization target. An antisense oligomer is specific hybridization when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA product, and when there is a sufficient degree of complementarity to prevent non-specific binding of the antisense oligomer to non-target sequences under the conditions of desired specific binding (i.e., the physiological conditions under which in vivo assays or therapeutic treatments and in vitro assays are performed, and the conditions under which these assays are performed).

[0098] While selective hybridization can be performed under low, medium, or high stringency conditions, high stringency conditions are preferred. Those skilled in the art will recognize that, in addition to base composition, complementary strand length, and the number of nucleotide base mismatches between hybridized nucleic acids, the stringency of hybridization will also be affected by conditions such as salt concentration, temperature, or organic solvents. Stringent temperature conditions will typically include temperatures exceeding 30°C, typically exceeding 37°C, preferably exceeding 45°C, preferably at least 50°C, and typically 60°C–80°C or higher. Stringent salt conditions will typically be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is more important than the measurement of any single parameter. An example of stringent hybridization conditions is 65°C and 0.1xSSC (1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Therefore, the antisense oligomers of the present invention may include oligomers that selectively hybridize with the sequences (SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41) provided in Table 1.

[0099] At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes with the complementary polynucleotide. This hybridization may occur when the antisense oligomer has “near” or “significant” complementarity with the target sequence, as well as precise complementarity.

[0100] Typically, selective hybridization occurs when the nucleotides of the antisense oligomer have at least about 55% identity with the nucleotides of the antisense oligomer in a segment of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98%, or 99%. As described, the length of the homology comparison can be a longer segment, and in some embodiments it is typically in a segment of at least about nine nucleotides, typically at least about 12 nucleotides, more typically at least about 20 nucleotides, typically at least about 21, 22, 23, or 24 nucleotides, at least about 25, 26, 27, or 28 nucleotides, at least about 29, 30, 31, or 32 nucleotides, or at least about 36 or more nucleotides.

[0101] Therefore, the antisense oligomer sequence of the present invention preferably has at least 75%, more preferably at least 85%, more preferably at least 86%, 87%, 88%, 89%, or 90% homology with the sequence listed herein. More preferably, it has at least 91%, 92%, 93%, 94%, or 95%, more preferably at least 96%, 97%, 98%, or 99% homology. Generally, the shorter the length of the antisense oligomer, the higher the homology required to obtain selective hybridization. Therefore, when the antisense oligomer of the present invention consists of fewer than about 30 nucleotides, it is preferred that the percentage of identity with the antisense oligomers listed herein is greater than 75%, preferably greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Nucleotide homology comparisons can be performed using sequence comparison programs such as GCG Wisconsin Bestfit or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or significantly different lengths to those cited herein can be compared by inserting vacancies into the alignment; such vacancies are identified, for example, by the comparison algorithm used in GAP.

[0102] The antisense oligomers of the present invention can have regions of reduced homology and regions of precise homology with the target sequence. The oligomer does not necessarily need to have precise homology along its entire length. For example, the oligomer can have a continuous segment of at least 4 or 5 bases identical to the target sequence, preferably a continuous segment of at least 6 or 7 bases identical to the target sequence, more preferably a continuous segment of at least 8 or 9 bases identical to the target sequence. The oligomer can have a segment of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases identical to the target sequence. The remaining segments of the oligomer sequence can be intermittently identical to the target sequence; for example, the remaining sequence can have the same bases, followed by different bases, and then the same bases again. Alternatively (or additionally), the oligomeric sequence may have several identical sequences (e.g., 3, 4, 5, or 6 bases) interspersed with incompletely homologous segments. Such sequence mismatches will preferably result in little or no loss of cleavage modification activity.

[0103] The term “regulation” includes an optionally defined and / or statistically significant amount that “increases” or “decreases” one or more quantifiable parameters. The terms “increase,” “enhance,” or “stimulate” generally refer to the ability of one or more antisense oligomers or compositions to produce or induce a greater physiological response (i.e., downstream effect) in cells or subjects relative to a response not induced by the antisense oligomer or by a control compound.

[0104] The terms "enhancement," "increase," or "stimulation" generally refer to the ability of one or more antisense compounds or compositions to produce or evoke a greater physiological response (i.e., downstream effect) in cells or a subject compared to a response not induced by the antisense compound or by a control compound. Measurable physiological responses may include increased expression of the functional form of the NEAT1 protein, as well as other responses that are apparent from the understanding in the art and the description herein. The amount of "increase" or "enhancement" is generally a "statistically significant" amount and may include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more times (e.g., 500, 1000 times) compared to the amount produced by the antisense compound (in the absence of the reagent) or by the control compound (including all integers and decimals in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0105] The term “reduction” generally refers to the ability of one or more antisense oligomers or compositions to produce or induce a smaller physiological response (i.e., downstream effect) in cells or a subject, relative to a response not induced by the antisense oligomer or by a control compound. The terms “reduction” or “inhibition” can generally refer to the ability of one or more of the antisense compounds of this invention to “reduce” a relevant physiological or cellular response (such as symptoms of the disease or condition described herein, as measured according to conventional techniques in the diagnostic field). Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in symptoms or signs of PTP1B-related conditions. The “reduction” in the reaction compared to a reaction not produced by an antisense compound or by a control composition can be statistically significant and can include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers in between.

[0106] The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the amount of PTP1B protein. An “increased” or “enhanced” amount is generally a statistically significant amount and may include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more times (e.g., 500, 1000 times) (inclusive of all integers and decimals above 1, e.g., 1.5, 1.6, 1.7, 1.8) compared to the amount produced by the antisense oligomer (in the absence of the reagent) or by the control compound. The terms “reduced” or “inhibited” may generally refer to the ability of one or more antisense oligomers to “reduce” the relevant physiological or cellular response (such as the symptoms of the disease or condition described herein, as measured according to conventional techniques in the diagnostic field). The relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in symptoms or signs of diseases associated with T2DM (such as insulin resistance and leptin resistance) or diseases such as cancer. The “reduction” in response compared to a response not produced by the antisense oligomer or by the control composition may be statistically significant and may include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers in between.

[0107] The length of the antisense oligomer can vary, as long as it can selectively bind to the desired location within the precursor mRNA molecule. The length of such sequences can be determined according to the selection procedure described herein. Typically, the length of the antisense oligomer will be from about 10 nucleotides to about 50 nucleotides. However, it will be understood that nucleotides of any length within this range can be used in this method. Preferably, the length of the antisense oligomer is between 10 and 40, 10 and 35, 15 and 30 nucleotides, or 20 and 30 nucleotides, most preferably about 25 and 30 nucleotides. For example, the length of the oligomer can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0108] As used herein, “antisense oligomer” or “ASO” refers to a linear sequence of a nucleotide or nucleotide analog that allows nucleobases to hybridize with a target sequence in RNA via Watson-Crick base pairing to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms “antisense oligomer,” “antisense oligonucleotide,” “oligomer,” and “antisense compound” are used interchangeably to refer to oligonucleotides. The cyclic subunit may be based on ribose or another pentose, or in some embodiments, on a morpholino group (see description of morpholino oligonucleotides below). Peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl (2'-OMe) oligonucleotides, as well as other antisense agents known in the art, are also considered.

[0109] In some implementations, the antisense oligonucleotide has the chemical composition of a naturally occurring nucleic acid molecule, that is, the antisense oligonucleotide does not include modified or substituted bases, sugars, or inter-subunit bonds.

[0110] In a preferred embodiment, the antisense oligonucleotide of the present invention is a non-naturally occurring nucleic acid molecule or an "oligonucleotide analog". For example, a non-naturally occurring nucleic acid may include one or more non-natural bases, sugars, and / or inter-subunit bonds, for example, bases, sugars, and / or bonds that have been modified or substituted relative to bases, sugars, and / or bonds present in naturally occurring nucleic acid molecules. Exemplary modifications are described below. In some embodiments, the non-naturally occurring nucleic acid includes more than one type of modification, such as sugar and base modification, sugar and bond modification, base and bond modification, or base, sugar, and bond modification. For example, in some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) bases. In some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) sugars. In some embodiments, the antisense oligonucleotide contains non-natural (e.g., modified or substituted) inter-subunit bonds. In some embodiments, the antisense oligonucleotide contains more than one type of modification or substitution, such as non-natural bases and / or non-natural sugars and / or non-natural inter-subunit bonds.

[0111] Therefore, non-naturally occurring antisense oligomers are included, which possess the following characteristics: (i) a modified backbone structure, for example, a backbone other than the standard phosphodiester bonds present in naturally occurring oligonucleotides and polynucleotides, and / or (ii) a modified sugar moiety, for example, a morpholino moiety instead of a ribose or deoxyribose moiety. Oligonucleotide analogs support bases capable of hydrogen bonding with standard polynucleotide bases via Watson-Crick base pairing, wherein the analog backbone presents bases in a manner that allows for sequence-specific hydrogen bonding between the oligonucleotide analog molecule and the bases of the standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphorus-containing backbone.

[0112] One method for generating antisense oligomers is the methylation of the 2' hydroxyribose position, and the incorporation of a thiophosphate backbone produces molecules that are surface-like with RNA but more resistant to nuclease degradation. However, those skilled in the art will recognize other suitable backbones that can be used for the purposes of this invention.

[0113] To avoid precursor RNA degradation during duplex formation with antisense oligomers, the antisense oligomers used in this method can be modified to minimize or prevent cleavage by endogenous RNase H. Antisense molecules that do not activate RNase H can be prepared according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense molecules can be deoxyribonucleotide or ribonucleotide sequences, simply containing any structural modification that spatially hinders or prevents the binding of RNase H to the duplex molecule containing an oligonucleotide as a member of the duplex molecule, wherein the structural modification substantially does not hinder or disrupt duplex formation. Since the portions of the oligonucleotide involved in duplex formation are substantially different from those involved in the binding of RNase H, a wide variety of antisense molecules that do not activate RNase H can be used. This characteristic is highly preferred because treatment of RNA with unmethylated oligomers, whether intracellular or in crude extracts containing RNase H, leads to the degradation of the precursor mRNA:antisense oligomer duplex. Any form of modified oligomer capable of bypassing or not inducing such degradation can be used in this invention. Nuclease resistance can be achieved by modifying the antisense oligomer of this invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups (including carboxylic acid groups, ester groups, and alcohol groups).

[0114] An example of an antisense oligomer that is not cleaved by cellular RNase H when forming a duplex with RNA is a 2'-O-methyl derivative. Such 2'-O-methyl oligonucleotides are stable in cellular environments and animal tissues, and their duplexes with RNA have a higher Tm value than their ribose or deoxyribose counterparts. Alternatively, the nuclease-resistant antisense oligomers of the present invention may have at least one fluorinated final 3'-terminal nucleotide. Still alternatively, the nuclease-resistant antisense oligomers of the present invention have phosphate thioester bonds linked between at least two final 3'-terminal nucleotide bases, preferably phosphate thioester bonds linked between the final four 3'-terminal nucleotide bases.

[0115] Modified or regulated RNA splicing can also be achieved using alternative oligonucleotide chemical processes (see, for example, U.S. Patent No. 5,149,797). For example, antisense oligomers may be selected from a list including: aminophosphate or phosphatidylmorpholino oligomers (PMO); PMO-X; PPMO; peptide nucleic acids (PNA); locked nucleic acids (LNA) and derivatives, including α-L-LNA, 2'-aminoLNA, 4'-methylLNA and 4'-O-methylLNA; ethylidene-bridged nucleic acids (ENA) and their derivatives; thiophosphate oligomers; tricyclic DNA oligomers (tcDNA); tricyclic thiophosphate oligomers; 2'-O-methyl-modified oligomers (2'-OMe); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro, 2'-fluoroarabinose (FANA); unlocked nucleic acids (UNA); hexitol nucleic acids (HNA); cyclohexenyl nucleic acids (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine; or any combination of the above as a mixture or as a gapmer.

[0116] To further improve delivery efficiency, the modified nucleotides described above are typically conjugated to sugar or nucleobase moieties with fatty acids / lipids / cholesterols / amino acids / carbohydrates / polysaccharides / nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct antisense oligomers to modify cleavage factor binding. Antisense oligomer-induced splicing factor binding modification of the PTPN1 gene transcript typically uses bases on a phosphate thioester backbone modified with oligonucleotides, PNA, 2'OMe, or MOE. Although 2'OMe ASO is used in oligonucleotide design, it is not considered ideal for in vivo or clinical applications due to its high in vitro uptake efficiency as a cationic liposome and its sensitivity to nuclease degradation. When using alternative chemical processes to generate the antisense oligomers of this invention, the uracil (U) in the sequence provided herein can be replaced with thymine (T).

[0117] For example, such antisense molecules can be oligonucleotides in which at least one or all of the nucleotide-bridging phosphate residues are modified phosphate esters, such as methylphosphonates, methylthiophosphates, phosphoromorpholidates, phosphoropiperazidates, and phosphor amidates. For example, the phosphate ester bridging residues can be modified as described, for example, every other nucleotide. In another non-limiting example, such antisense molecules are molecules in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, the phosphate ester bridging residues can be modified as described, for example, every other nucleotide.

[0118] Specific examples of antisense oligonucleotides that can be used in this invention include oligonucleotides containing a modified backbone or non-natural subunit bonds.

[0119] Oligonucleotides with modified backbones include those oligonucleotides that retain phosphorus atoms in their backbone and those that do not. Modified oligonucleotides that do not have phosphorus atoms in their internucleotide backbone can also be considered oligonucleotides.

[0120] In other antisense molecules, both the sugar and nucleotide intermolecular bonds (i.e., the backbone of the nucleotide unit) are replaced by novel groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomer (an oligonucleotide mimic with excellent hybridization properties has been shown) is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and linked directly or indirectly to the nitrogen atom of the amide moiety of the backbone.

[0121] Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (commonly referred to in the art simply as "bases") modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more purine or pyrimidine bases most common in the nucleic acid are replaced by less common or non-natural bases.

[0122] Purine bases consist of a pyrimidine ring fused to an imidazole ring; adenine and guanine are the two most common purine nucleobases in nucleic acids. These can be substituted by other naturally occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0123] Pyrimidine bases contain a six-membered pyrimidine ring; cytosine, uracil, and thymine are the most common pyrimidine bases in nucleic acids. These can be substituted by other naturally occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotide described herein contains a thymine base in place of uracil.

[0124] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymidine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazoguanine, 7-deazoadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, Super G, Super A and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil, or derivatives thereof; and degenerate or universal bases, such as 2,6-difluorotoluene, or the absence of bases, such as abase sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives wherein the epoxide has been replaced by nitrogen (azaribose)). Examples of derivatives of Super A, Super G, and Super T can be found in U.S. Patent 6,683,173 (Epoch Biosciences). When incorporated into siRNA, cPent-G, cPent-AP, and Pr-AP exhibit reduced immunostimulatory effects (Peacock H et al., J. Am. Chem. Soc. 2011 133, 9200). Pseudorabyl is a naturally occurring isomerized version of uracil, containing a C-glycoside instead of the conventional N-glycoside found in uridine. Pseudorabyl-containing mRNAs may offer improved safety profiles compared to uridine-containing mPvNAs (see WO 2009127230).

[0125] Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine), 5-propynyluracil, and 5-propynylcytosine. Even more particularly, when combined with 2'-O-methoxyethyl sugar modification, 5-methylcytosine substitution shows an increase in the stability of the nucleic acid duplex by 0.6-1.2 °C and is currently the preferred base substitution.

[0126] In some embodiments, modified or substituted nucleobases can be used to facilitate the purification of antisense oligonucleotides. For example, in some embodiments, the antisense oligonucleotide may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In some antisense oligonucleotides, a string of three or more consecutive guanine bases can lead to oligonucleotide aggregation, complicating purification. In such antisense oligonucleotides, one or more of the consecutive guanine bases can be substituted with inosine. Replacing one or more guanine bases in a string of three or more consecutive guanine bases with inosine can reduce the aggregation of antisense oligonucleotides, thereby facilitating purification.

[0127] In one embodiment, another modification of the antisense oligonucleotide involves chemically linking one or more portions or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide to the oligonucleotide. Such portions include, but are not limited to, lipid portions (such as cholesterol portions), bile acids, thioethers (e.g., hexyl-5-triphenylmethanethiol, thiocholesterol), fatty chains (e.g., dodecyldiol or undecyl residues), phospholipids (e.g., di-hexadecyl-racemic-glycerol or triethylammonium-1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate), polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl portions, or octadecylamine or hexylamino-carbonyl-hydroxycholesterol portions.

[0128] It is not necessary to uniformly modify all positions in a given compound, and in fact, more than one of the above modifications can be incorporated into a single compound or even into a single nucleoside within an oligonucleotide. The invention also includes antisense oligonucleotides for chimeric compounds. In the context of this invention, a "chimeric" antisense compound or "chimera" is an antisense molecule (particularly an oligonucleotide) containing two or more chemically distinct regions, each region consisting of at least one monomeric unit (i.e., a nucleotide in the case of oligonucleotide compounds). These oligonucleotides typically contain at least one region in which the oligonucleotide is modified to increase resistance to nuclease degradation and cellular uptake, and an additional region for increasing binding affinity to target nucleic acids.

[0129] The antisense molecules used according to the present invention can be conveniently and conventionally prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). U.S. Patent No. 4,458,066 describes a method for synthesizing oligonucleotides on a modified solid support.

[0130] In another non-limiting example, such an antisense oligomer is a molecule in which at least one or all of its nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, modifications can be made every other nucleotide as described.

[0131] While the above-described antisense oligomers are preferred forms of antisense oligomers of the present invention, the present invention includes other oligomeric antisense molecules, including but not limited to the oligomeric mimics described below.

[0132] Another preferred chemical is the phosphatidylmorpholino oligomer (PMO) compound, which is not degraded by any known nuclease or protease. These compounds are uncharged, do not activate RNase H activity when bound to the RNA chain, and have been shown to exert sustained cleavage factor binding regulation after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0133] The modified oligomers may also contain one or more substituted sugar moieties. The oligomers may also include nucleobase (generally referred to simply as "bases" in the art) modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine), 5-propynyluracil, and 5-propynylcytosine. Even more particularly, when combined with 2'-O-methoxyethyl sugar modifications, 5-methylcytosine substitution has been shown to increase the stability of the nucleic acid duplex by 0.6-1.2 °C. In one embodiment, at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, wherein the pyrimidine base is selected from the group consisting of cytosine, thymine, and uracil. In one embodiment, the 5-substituted pyrimidine base is 5-methylcytosine. In another embodiment, at least one purine base of the oligonucleotide comprises an N-2, N-6 substituted purine base. In one embodiment, the N-2, N-6 substituted purine base is 2,6-diaminopurine.

[0134] In one embodiment, the antisense oligonucleotide comprises one or more 5-methylcytosine substitutions, alone or in combination with another modification (such as a 2'-O-methoxyethyl sugar modification). In yet another embodiment, the antisense oligonucleotide comprises one or more 2,6-diaminopurine substitutions, alone or in combination with another modification.

[0135] In some embodiments, the antisense oligonucleotide is chemically linked to one or more portions (such as a polyethylene glycol portion) or conjugates (such as an arginine-rich cell-penetrating peptide) that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. In one exemplary embodiment, the arginine-rich polypeptide is covalently coupled at its N-terminal or C-terminal residue to the 3' or 5' end of the antisense compound. Similarly, in an exemplary embodiment, the antisense compound consists of an inter-unit bond between a morpholine subunit and a phosphorus-containing subunit, which links the morpholine nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit.

[0136] In another aspect, the present invention provides expression vectors incorporating the aforementioned antisense oligonucleotides (e.g., antisense oligonucleotides of SEQ ID NO: 1-41). In some embodiments, the expression vector is a modified retroviral or non-retroviral vector, such as an adeno-associated virus vector.

[0137] Another modification of the oligomers of the present invention involves chemically linking one or more portions or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligomer to the oligomer. Such portions include, but are not limited to, lipid portions (such as cholesterol portions), bile acids, thioethers (e.g., hexyl-S-triphenylmethanethiol, thiocholesterol), fatty acid chains (e.g., dodecyldiol or undecyl residues), phospholipids (e.g., di-hexadecyl-racemic-glycerol or triethylammonium-1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate), polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl portions, tetradecyl, or octadecylamine or hexylamino-carbonyl-hydroxycholesterol portions.

[0138] Cell-penetrating peptides have been added to phosphatidylmorpholino oligomers to enhance cellular uptake and nuclear localization. Different peptide tags have been shown to influence uptake efficiency and target tissue specificity, as illustrated in Jearawiriyapaisarn et al., (2008), Mol. Ther. 16 9, 1624-1629. The terms “cell-penetrating peptide” and “CPP” are used interchangeably and refer to cationic cell-penetrating peptides, also known as transport peptides, carrier peptides, or peptide transduction domains. As demonstrated herein, peptides have the ability to induce cell penetration within 100% of a given cell culture population and allow for macromolecular translocation in multiple tissues in vivo following systemic administration.

[0139] It is not necessary to uniformly modify all positions in a given compound, and in fact, more than one of the above modifications can be incorporated into a single compound or even into a single nucleoside within an oligomer. The invention also includes antisense oligomers for chimeric compounds. In the context of this invention, a "chimeric" antisense oligomer or "chimer" is an antisense oligomer (especially an oligomer) containing two or more chemically distinct regions, each region consisting of at least one monomeric unit (i.e., a nucleotide in the case of oligomeric compounds). These oligomers typically contain at least one region in which the oligomer is modified to increase resistance to nuclease degradation and cellular uptake, and an additional region for increasing binding affinity to target nucleic acids.

[0140] The activity of antisense oligomers and their variants can be determined using conventional techniques in the art. For example, the isoform and expression levels of identified RNA and protein can be assessed using any of a variety of well-known methods for detecting the isoform and / or expression of transcribed nucleic acids or proteins. Non-limiting examples of such methods include: RT-PCR of RNA isoforms followed by size separation of PCR products; nucleic acid hybridization methods, such as Northern blotting and / or the use of nucleic acid arrays; fluorescence in situ hybridization for detecting intracellular RNA transcription; nucleic acid amplification methods; immunological methods for detecting proteins; protein purification methods; and assays of protein function or activity.

[0141] RNA / cDNA (i.e., transcribed polynucleotides) can be prepared from cells, tissues, or organisms, and RNA expression levels can be assessed by hybridizing the RNA / cDNA with a reference polynucleotide, which is a complement of the nucleic acid being measured or a fragment thereof. cDNA can optionally be amplified using any of a variety of polymerase chain reaction or in vitro transcription methods prior to hybridization with the complementary polynucleotide; preferably, the cDNA is not amplified. Quantitative PCR can also be used to detect the expression of one or more transcripts to assess the expression level of transcript T1.

[0142] This invention provides antisense oligomers that modify the splicing factor binding of the PTPN1 gene transcript, clinically relevant oligomer chemistry processes, and delivery systems to directly reduce the full-length PTPN1 transcript to therapeutic levels. This is achieved through the following methods to substantially alter the amount of PTPN1 RNA:

[0143] 1) Oligomer purification was performed in vitro using cell lines by experimentally evaluating (i) modifications to splicing factors binding to target motifs, (ii) the development of antisense oligomer lengths and oligomer mixtures, (iii) the selection of chemical processes, and (iv) the addition of cell-penetrating peptides (CPPs) to enhance oligomer delivery; and

[0144] 2) Detailed evaluation of new protocols for reducing PTPN1 transcripts.

[0145] Therefore, this paper demonstrates that the processing of PTPN1 RNA can be manipulated using specific antisense oligomers. In this way, the function of a certain amount of PTP1B protein can be significantly reduced, thereby reducing the symptoms of diseases associated with PTP1B.

[0146] Preferably, the diseases associated with PTP1B are: (i) diseases associated with downregulation of insulin signaling in the subject; (ii) diseases associated with downregulation of the leptin signaling pathway in the subject; and / or (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the disease may be type 2 diabetes mellitus (T2DM) and / or obesity. Additionally, the disease may be solid tumors.

[0147] The antisense oligomers used according to the present invention can be conveniently prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). U.S. Patent No. 4,458,066 describes a method for synthesizing oligomers on a modified solid support.

[0148] Any other means known in the art for such synthesis may be employed additionally or alternatively. Similar techniques are well known for the preparation of oligomers, such as thiophosphates and alkylated derivatives. In one such automated embodiment, diethylphosphamide is used as a starting material and can be synthesized as described by Beaucage et al., (1981) Tetrahedron Letters, 22: 1859-1862.

[0149] The antisense oligomers of the present invention are synthesized in vitro and do not include biologically derived antisense compositions or genetic vector constructs designed to guide the in vivo synthesis of antisense oligomers. The molecules of the present invention can also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds to form, for example, liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations to aid in uptake, distribution, and / or absorption.

[0150] This also includes vector delivery systems capable of expressing the oligo-NEAT1 targeting sequence of the present invention, such as vectors expressing polynucleotide sequences comprising any one or more of SEQ ID NOs: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41, as described herein. The term "vector" or "nucleic acid construct" means a polynucleotide molecule, preferably a DNA molecule, derived, for example, from a plasmid, bacteriophage, yeast, or virus in which polynucleotides can be inserted or cloned. The vector preferably contains one or more unique restriction sites and is capable of autonomous replication in a defined host cell, including a target cell or tissue or a progenitor cell or tissue thereof, or can integrate with the genome of a defined host such that the cloned sequence is reproducible. Therefore, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, for example, a linear or closed circular plasmid, an extrachromosomal element, a miniature chromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector can be one that, when introduced into a host cell, is integrated into the genome and replicates along with the chromosome into which it is integrated.

[0151] Treatment

[0152] The antisense oligomers of the present invention can also be used as preventative or therapeutic agents for the purpose of treating diseases. Therefore, in one embodiment, the present invention provides an antisense oligomer that binds to a selected target in PTPN1 RNA at a therapeutically effective amount to modify RNA splicing as described herein, which is mixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0153] "Effective dose" or "therapeutic effective dose" refers to the amount of a therapeutic compound (such as an antisense oligomer) that is administered to a mammalian subject as a single dose or as part of a series of doses and effectively produces the intended therapeutic effect.

[0154] Therefore, the present invention provides a pharmaceutical, preventive, or therapeutic composition for treating, preventing, or improving the effects of PTP1B-related diseases in a subject, the composition comprising:

[0155] a) one or more antisense oligomers as described herein, and

[0156] b) One or more drug-acceptable carriers and / or diluents.

[0157] Preferably, the diseases associated with PTP1B are: (i) diseases associated with downregulation of insulin signaling in the subject; (ii) diseases associated with downregulation of the leptin signaling pathway in the subject; and / or (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the diseases may be type 2 diabetes mellitus (T2DM), obesity, or solid tumors.

[0158] Preferably, the antisense oligomers used in this invention are selected from a list including the following:

[0159] ·SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41;

[0160] • SEQ ID NO: 1 or 32-36; or

[0161] •SEQ ID NO: 33.

[0162] Preferably, the antisense oligomer causes exon hopping in exon 2.

[0163] The composition may comprise about 1 nM to 1000 nM of each desired antisense oligomer of the present invention. Preferably, the composition may comprise about 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 10 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, and most preferably 1 nM to 10 nM of each antisense oligomer of the present invention.

[0164] The composition may comprise each of the desired antisense oligomers of the present invention at a wavelength of about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm.

[0165] The present invention further provides one or more antisense oligomers adapted to assist in preventive or therapeutic treatment, prevention or improvement of symptoms of diseases or conditions associated with PTP1B in a form suitable for delivery to a subject.

[0166] The phrase “pharmaceutical acceptable” refers to a molecular entity and composition that is physiologically tolerable and, when administered to a subject, generally does not produce allergic reactions or similar adverse reactions (such as stomach upset). The term “carrier” refers to a diluent, adjuvant, excipient, or transporter that is administered with a compound. Such drug carriers can be sterile liquids, such as water and oils, including those from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Aqueous or saline solutions, as well as aqueous glucose and glycerol solutions, are preferred as carriers, particularly injectable solutions. Suitable drug carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990).

[0167] Pharmaceutical Composition

[0168] In this invention, pharmaceutical compositions are provided comprising a therapeutically effective amount of one or more of the antisense oligomers of the invention, and pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include: diluents having various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80); antioxidants (e.g., ascorbic acid, sodium metabisulfite); preservatives (e.g., thimerosal, benzyl alcohol); and bulking agents (e.g., lactose, mannitol). The materials can be incorporated into particulate formulations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, etc.) or into liposomes. Hyaluronic acid can also be used. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the invention. See, for example, Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712, which is incorporated herein by reference. The composition may be prepared in liquid form or in a dry powder form, such as a lyophilized form.

[0169] It will be understood that the pharmaceutical compositions provided according to the present invention can be administered by any means known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, oral administration, topical application, or via the lung or nose. More preferably, the antisense oligomers are delivered via intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable route can be determined by those skilled in the art based on the condition of the subject receiving treatment. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are some non-limiting sites in which the antisense oligomers can be introduced. Direct CNS delivery can be employed; for example, intraventricular or intrathecal administration can be used as a route of administration.

[0170] Formulations for topical application include those in which the oligomers of this disclosure are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine, dimyristoylphosphatidylcholine DMPC, distearate phosphatidylcholine), anion-dependent (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other applications, the oligomers of this disclosure may be encapsulated in liposomes or may form complexes with them (especially cationic liposomes). Alternatively, the oligomers may be complexed with lipids (especially cationic lipids). Fatty acids and esters, their pharmaceutically acceptable salts, and their uses are further described in U.S. Patent No. 6,287,860 and / or U.S. Patent Application Serial No. 09 / 315,298, filed May 20, 1999.

[0171] In some embodiments, the antisense oligomers of this disclosure can be delivered via transdermal methods (e.g., by incorporating the antisense oligomers into, for example, an emulsion, wherein such antisense oligomers are optionally packaged in liposomes). In the art (e.g., in U.S. Patent No. 6,965,025), such transdermal and emulsion / liposome-mediated delivery methods are described for delivering antisense oligomers.

[0172] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is a well-established process in the art, such as the synthetic implant design described in U.S. Patent No. 6,969,400.

[0173] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be desired. Oral formulations are those in which the oligomers of this disclosure are administered in combination with one or more penetration enhancers, surfactants, and chelating agents. Surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Bile acids / salts and fatty acids, and their uses, are further described in U.S. Patent No. 6,287,860. In some embodiments, this disclosure provides combinations of penetration enhancers, such as combinations of fatty acids / salts with bile acids / salts. Exemplary combinations are sodium salts of dodecanoic acid, decanoic acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-dodecyl ether and polyoxyethylene-20-hexadecyl ether. The oligomers disclosed herein can be delivered orally in granular form, including spray-dried granules, or compounded to form microparticles or nanoparticles. Oligomeric compound formulations and their uses are further described in U.S. Patent No. 6,287,860. Oral oligomeric formulations and their preparation are described in detail in US 6,887,906, 09 / 315,298 (filed May 20, 1999), and / or US 20030027780.

[0174] Compositions and formulations intended for parenteral, intrathecal, or intracardiac administration may include sterile aqueous solutions and may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0175] The delivery of therapeutically useful amounts of antisense oligomers can be achieved through previously disclosed methods. For example, antisense oligomers can be delivered intracellularly through a composition comprising a mixture of the antisense oligomer and an effective amount of a block copolymer. Examples of this method are described in U.S. Patent Application US20040248833. Other methods for delivering antisense oligomers to the cell nucleus are described in Mann CJ et al., (2001) Proc, Natl. Acad. Science, 98(1): 42-47 and in Gebski et al., (2003) Human Molecular Genetics, 12(15): 1801-1811. US 6,806,084 describes a method for introducing nucleic acid molecules into cells by using an expression vector as naked DNA or conjugated with a lipid carrier.

[0176] In some embodiments, the antisense oligomers of the present invention and therapeutic compositions comprising them can be delivered via transdermal methods (e.g., by incorporating the antisense oligomers into, for example, an emulsion, wherein such antisense oligomers are optionally packaged in liposomes). In the art (e.g., in U.S. Patent No. 6,965,025), such transdermal and emulsion / liposome-mediated delivery methods are described for delivering antisense oligomers.

[0177] Delivery of antisense oligomers may be desired in colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations. These colloidal dispersion systems can be used to manufacture therapeutic pharmaceutical compositions.

[0178] Liposomes are artificial membrane vesicles suitable for use as delivery carriers in vitro and in vivo. These formulations can be characterized by net cations, anions, or neutral charges, and possess useful features for in vitro, in vivo, and ex vivo delivery methods. It has been shown that large monolayer vesicles can encapsulate large amounts of aqueous buffer solutions containing macromolecules. RNA and DNA can be encapsulated within aqueous solutions and delivered to cells in their biologically active form (Fraley et al., Trends Biochem. Sci. 677, 1981).

[0179] To make liposomes effective gene transfer vectors, they should exhibit the following characteristics: (1) efficient encapsulation of antisense oligomers of interest without impairing their biological activity; (2) preferential binding to target cells over non-target cells; (3) efficient delivery of the aqueous contents of the vesicles to the cytoplasm of the target cells; and (4) accurate and efficient expression of genetic information (Mannino et al., Biotechniques, 6:682, 1988). Liposome combinations are typically combinations of phospholipids (especially those with high phase transition temperatures), often combined with steroids (especially cholesterol). Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to retain DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA to cells.

[0180] Liposomes also include “sterically stable” liposomes, as used herein; this term refers to liposomes containing one or more specialized lipids that, when incorporated into liposomes, result in increased cycle life compared to liposomes lacking such specialized lipids. Examples of sterically stable liposomes are those in which a portion of the lipid portion forming the vesicle contains one or more glycolipids or is derived from one or more hydrophilic polymers, such as a polyethylene glycol (PEG) portion. Liposomes and their uses are further described in US 6,287,860.

[0181] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is a well-established process in the art, such as the synthetic implant design described in U.S. Patent No. 6,969,400, the disclosure of which is incorporated herein by reference in its entirety.

[0182] Antisense oligomers can be introduced into cells using techniques well-established in the art, such as transfection, electroporation, fusion, liposomes, colloidal polymer particles and viral and nonviral vectors, and other methods known in the art. The chosen delivery method will depend at least on the cells to be treated and their location, and will be readily apparent to a skilled technician. For example, localization can be achieved by liposomes with specific markers on their surface for guiding the liposomes, direct injection into tissues containing target cells, or specific receptor-mediated uptake.

[0183] As is known in the art, antisense oligomers can be delivered using methods involving, for example, liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake and receptor-mediated endocytosis, as well as additional non-endocytic delivery modalities such as microinjection, permeation (e.g., streptococcal hemolysin-O permeation, anionic peptide permeation), electroporation and various non-invasive non-endocytic delivery methods known in the art (ref. Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, which are incorporated herein by reference in their entirety).

[0184] Antisense oligomers can also be combined with other pharmaceutically acceptable carriers or diluents to produce pharmaceutical compositions. Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0185] The described routes of administration are intended as guidance only, as skilled practitioners will be able to easily determine the optimal route of administration and any dosage for any particular animal and condition.

[0186] Various approaches have been explored for introducing novel functional genetic material into cells in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include integrating the gene to be expressed into a modified retrovirus (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.:5074S-5079S); integrating it into a non-retroviral vector (Rosenfeld et al., (1992) Cell, 68:143-155; Rosenfeld et al., (1991) Science, 252:431-434); or delivering transgenes linked to heterologous promoter-enhancer elements via liposomes (Friedmann (1989) supra; Brigham et al., (1989) Am.J.Med.Sci., 298:278-281; ​​Nabel et al., (1990) Science, 249:1285-1288; Hazinski et al., (1991) Am.J.Resp.Cell). Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624); or using naked DNA, expression vectors (Nabel et al., (1990), ibid.); Wolff et al., (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues only produces local expression (Rosenfeld (1992), ibid.); Rosenfeld et al., (1991), ibid.); Brigham et al., (1989), ibid.; Nabel (1990), ibid.; and Hazinski et al., (1991), ibid.). The Brigham et al. group (Am.J.Med.Sci.(1989)298:278-281 and Clinical Research(1991)39(abstract)) have reported that in vivo transfection was performed only in the lungs of mice after intravenous or intratracheal administration of DNA liposome complexes.Examples of review articles on human gene therapy procedures include: Anderson, Science (1992) 256:808-813; Barteau et al., (2008), Curr Gene Ther; 8(5):313-23; Mueller et al., (2008). Clin Rev Allergy Immunol; 35(3):164-78; Li et al., (2006) Gene Ther., 13(18):1313-9; Simoes et al., (2005) Expert Opin Drug Deliv; 2(2):237-54.

[0187] The antisense oligomers of the present invention encompass any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound that, when administered to animals, including humans, is capable of (directly or indirectly) providing a bioactive metabolite or its residue. Therefore, by way of example, this disclosure also relates to prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0188] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compounds of the present invention: that is, a salt that retains the desired biological activity of the parent compound without conferring undesirable toxicological effects. Preferred examples of pharmaceutically acceptable salts for oligomers include, but are not limited to, (a) salts formed with cations (such as sodium, potassium, ammonium, magnesium, calcium, polyamines (such as spermine and spermidine)); (b) acid addition salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.); (c) salts formed with organic acids (such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.); and (d) salts formed by elemental anions (such as chlorine, bromine, and iodine). The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ocular and mucous membrane delivery, and rectal delivery), pulmonary (e.g., by inhalation or blowing of powders or aerosols (including via nebulizer, intratracheal, intranasal, epidermal, and transdermal delivery)), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial administration, for example, intrathecal or intraventricular administration. Oligomers having at least one 2'-O-methoxyethyl modification are considered particularly suitable for oral administration. Preferably, antisense oligomers are delivered via subcutaneous or intravenous routes.

[0189] The pharmaceutical formulations of the present invention can be conveniently presented in unit dosage forms and can be prepared according to conventional techniques well-known in the pharmaceutical industry. Such techniques include the step of combining an active ingredient with a pharmaceutical carrier or excipient. Generally, the formulation is prepared by uniformly and closely combining the active ingredient with a liquid carrier or a finely dispersed solid carrier, or both, and then, if desired, shaping the product.

[0190] application

[0191] In one implementation, the antisense oligomer is administered in an amount and manner that effectively results in a peak blood concentration of at least 200-400 nM. Typically, one or more doses of the antisense oligomer are administered at regular intervals for approximately one to two weeks. The preferred dose for oral administration is approximately 1 mg to 1000 mg of oligomer per 70 kg. In some cases, a dose greater than 1000 mg of oligomer per subject may be required. For intravenous administration, the preferred dose is approximately 0.5 mg to 1000 mg of oligomer per 70 kg. For intravenous or subcutaneous administration, the antisense oligomer may be administered at a dose of approximately 120 mg / kg daily or weekly.

[0192] Antisense oligomers can be administered at regular intervals for short periods of time, such as daily for two weeks or less. However, in some cases, oligomers are administered intermittently over longer periods. They can be administered before or concurrently with antibiotics or other therapeutic treatments. Treatment regimens (dosage, frequency, route, etc.) can be adjusted as indicated based on the results of immunoassays, other biochemical tests, and physiological examinations of the treated subject.

[0193] Dosing depends on the severity and responsiveness of the disease state being treated, with the treatment process lasting from several days to several months, or until a cure or reduction of the disease state is achieved. The optimal dosing regimen can be calculated based on measurements of drug accumulation in the subject's body. Those skilled in the art can readily determine the optimal dosage, method of administration, and repetition rate. The optimal dosage can vary based on the relative potency of individual oligomers and can generally be estimated based on the effective EC50 found in in vitro and in vivo animal models. Generally, the dose is from 0.01 μg to 100 g per kg body weight and can be administered once or more daily, weekly, monthly, or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the repetition rate of dosing based on measured residence time and concentration of the drug in body fluids or tissues. After successful treatment, it may be necessary to subject the subject to maintenance therapy to prevent relapse of the disease state, wherein the oligomer is administered at a maintenance dose ranging from 0.01 μg to 100 g per kg body weight, once or more daily to once every 20 years.

[0194] Effective in vivo treatment regimens using the antisense oligomers of this invention can vary depending on the duration, dosage, frequency, and route of administration, as well as the condition of the treated subject (i.e., prophylactic administration versus administration in response to local or systemic infection). Therefore, such in vivo therapies typically require monitoring through trials appropriate to the specific type of condition for treatment, and corresponding adjustments to the dosage or treatment regimen to achieve optimal therapeutic outcomes.

[0195] Treatment can be monitored, for example, by general indicators of diseases known in the art. As used herein, “treatment” for a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural processes of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered preventively or after the onset of a pathological event or at the time of exposure to a pathogen. Treatment includes any desired effect on symptoms or signs of a disease or condition associated with PTP1B and may include, for example, minimal change or improvement in one or more measurable markers of a disease or condition being treated. “Preventative” treatment is also included, which may involve reducing the rate of progression of a disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily indicate the complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0196] As used herein, "subject" includes any animal that exhibits symptoms, is at risk of exhibiting such symptoms, or exhibits any symptoms associated with these conditions, which can be treated with the antisense compounds of the present invention (e.g., downregulating insulin signaling, downregulating the leptin signaling pathway, reducing cancer cell growth, migration, and invasion). Suitable subjects include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates and preferably human subjects are also included.

[0197] The efficacy of the antisense oligomers of the present invention administered in vivo can be determined based on biological samples (tissues, blood, urine, etc.) taken from the subject before, during, and after administration of the antisense oligomers. Measurements of such samples include (1) monitoring for the presence or absence of heteroduplexes with target and non-target sequences using procedures known to those skilled in the art (e.g., electrophoretic gel mobility assays); and (2) monitoring for the amount of mutant RNA associated with a reference normal RNA or protein, as determined by standard techniques (e.g., RT-PCR, Northern blotting, ELISA, or Western blotting).

[0198] For antisense oligomers, intranuclear oligomer delivery is a major challenge. Different cell-penetrating peptides (CPPs) localize PMO to varying degrees under different conditions and in different cell lines, and the inventors have evaluated the ability of novel CPPs to deliver PMO to target cells. The term CPP or “peptide moiety that enhances cellular uptake” is used interchangeably and refers to a cationic cell-penetrating peptide, also known as a “transport peptide,” “carrier peptide,” or “peptide transduction domain.” As shown herein, peptides have the ability to induce cell penetration within about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a given cell culture population and allow for the translocation of macromolecules in multiple tissues in vivo after systemic administration. CPPs are well known in the art and are disclosed, for example, in U.S. Application No. 2010 / 0016215, which is incorporated herein by reference in its entirety.

[0199] Therefore, the present invention provides a combination of the antisense oligomer of the present invention and a cell-penetrating peptide for use in the manufacture of therapeutic pharmaceutical compositions.

[0200] According to a further aspect of the invention, one or more antisense oligomers as described herein are provided for use in antisense oligomer-based therapies. Preferably, the therapy targets a disease associated with PTP1B.

[0201] Preferably, the diseases associated with PTP1B are: (i) diseases associated with downregulation of insulin signaling in the subject; (ii) diseases associated with downregulation of the leptin signaling pathway in the subject; and / or (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the disease may be type 2 diabetes mellitus (T2DM) and / or obesity. Alternatively, the disease may be solid tumors.

[0202] More specifically, the antisense oligomer is selected from the group consisting of any one or more of SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 and combinations thereof. This includes sequences that can hybridize with such sequences under strict hybridization conditions, sequences complementary to such sequences, sequences containing modified bases, sequences with modified backbones, and their functional truncations or extensions that control or regulate precursor RNA processing activity in the PTPN1 gene transcript. More preferably, the antisense oligomer used in this invention is selected from the list comprising: SEQ ID No: 1 or 32-36. Most preferably, the antisense oligomer used in this invention is SEQ ID NO: 33. Preferably, the antisense oligomer causes exon skipping in exon 2.

[0203] The invention also extends to combinations of two or more antisense oligomers capable of binding to selected targets to modify the splicing of the PTPN1 gene transcript. This combination can be a mixture of two or more antisense oligomers, or a construct comprising two or more antisense oligomers linked together for use in antisense oligomer-based therapies.

[0204] This invention provides a method for treating, preventing, or improving the effects of diseases associated with PTP1B, the method comprising the following steps:

[0205] a) Administering an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers as described herein to a subject.

[0206] Furthermore, the present invention provides a method for treating, preventing, or improving diseases associated with PTP1B, the method comprising the following steps:

[0207] a) Administering an effective amount of one or more antisense oligomers or pharmaceutical compositions, the pharmaceutical compositions comprising one or more antisense oligomers as described herein, to a subject.

[0208] The diseases associated with PTP1B include: (i) diseases associated with downregulation of insulin signaling in subjects; (ii) diseases associated with downregulation of the leptin signaling pathway in subjects; and (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the disease could be type 2 diabetes mellitus (T2DM) and / or obesity. Alternatively, the disease could be solid tumors.

[0209] Preferably, the therapy is used to develop nonfunctional, truncated, or nonsense PTP1B proteins. Preferably, the reduction of PTP1B levels is achieved by reducing the level of the full-length transcript through exon skipping achieved by binding to splice sites and / or by modifying the binding of precursor mRNA splicing factors in the PTPN1 gene transcript or a portion thereof.

[0210] A reduction in PTP1B will preferably result in a reduction in the number, duration, or severity of disease symptoms associated with: (i) downregulation of insulin signaling; (ii) downregulation of leptin signaling pathways, such as T2DM and / or obesity; and / or (iii) a reduction in cancer cell growth, migration, and invasion.

[0211] According to another aspect of the invention, the use of one or more antisense oligomers as described herein in the preparation of medicaments for regulating or controlling diseases associated with PTP1B is provided.

[0212] The present invention also provides the use of the purified and isolated antisense oligomers as described herein for the preparation of a medicament for the treatment of diseases associated with PTP1B.

[0213] The use of purified and isolated antisense oligomers, as described herein, for the preparation of medicaments for the treatment, prevention, or improvement of the effects of diseases associated with PTP1B is provided.

[0214] Preferably, the antisense oligomers used in this invention are selected from a list including the following:

[0215] ·SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41;

[0216] • SEQ ID NO: 1 or 32-36; or

[0217] •SEQ ID NO: 33.

[0218] Preferably, the antisense oligomer causes exon hopping in exon 2.

[0219] According to a further aspect of the invention, the invention extends to cDNA or clonal copies of the antisense oligomer sequence of the invention, and vectors containing the antisense oligomer sequence of the invention. The invention also extends further to cells containing such sequences and / or vectors.

[0220] The present invention also provides a kit for treating, preventing or improving diseases associated with PTP1B in subjects, the kit comprising at least isolated or purified antisense oligomers packaged in a suitable container, and instructions for use, the antisense oligomers being used to modify the binding of precursor mRNA splicing factors in the PTPN1 gene transcript or a portion thereof.

[0221] In a preferred embodiment, the kit contains at least one antisense oligomer as described herein or as shown in Table 1 (SEQ ID NO: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41), or a mixture of antisense oligomers as described herein. The kit may also contain peripheral reagents, such as buffers, stabilizers, etc.

[0222] Therefore, a kit is provided for the treatment, prevention or improvement of PTP1B-associated disease in a subject, the kit comprising at least an antisense oligomer as described herein or SEQ ID NOs 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 as shown in Table 1, and combinations or mixtures thereof, and instructions for use.

[0223] Also provided is a kit for treating, preventing or improving a disease associated with PTP1B in a subject, the kit comprising at least an antisense oligomer packaged in a suitable container, and instructions for use, the antisense oligomer being selected from the group consisting of any one or more of SEQ ID Nos: 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 and combinations thereof.

[0224] Preferably, the diseases associated with PTP1B are: (i) diseases associated with downregulation of insulin signaling in the subject; (ii) diseases associated with downregulation of the leptin signaling pathway in the subject; and / or (iii) diseases associated with cancer cell growth, migration, and invasion. For example, the disease may be type 2 diabetes mellitus (T2DM) and / or obesity. Alternatively, the disease may be solid tumors.

[0225] The contents of the kit may be lyophilized, and the kit may additionally contain a suitable solvent for reconstitution of the lyophilized components. The individual components of the kit will be packaged in separate containers, and accompanying such containers may be a notification in the form prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for manufacture, use, or sale for human administration.

[0226] When the components of the kit are provided in one or more liquid solutions, the liquid solutions may be aqueous solutions, such as sterile aqueous solutions. For in vivo use, the expression construct may be formulated into a pharmaceutically acceptable injectable composition. In this case, the container device itself may be an inhaler, syringe, pipette, eye dropper, or other similar device through which the formulation can be applied to the affected area (e.g., lung) of an animal, injected into the animal, or even applied to and mixed with other components of the kit.

[0227] The kit components may also be provided in dry or lyophilized form. When reagents or components are provided in dry form, they are typically reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container. Regardless of the number or type of containers, the kit of the present invention may also include or be packaged with tools for assisting in the injection / administration or placement of the final composite composition into an animal. Such tools may be inhalers, syringes, pipettes, forceps, measuring spoons, eye drops, or any such medically approved delivery medium.

[0228] Those skilled in the art will understand that the application of the above methods has wide applications in identifying antisense oligomers suitable for treating many other diseases.

[0229] The antisense oligomers of the present invention can also be used in combination with alternative therapies (such as drug therapy).

[0230] Therefore, the present invention provides a method for treating, preventing or improving the effects of a disease associated with PTP1B, wherein the antisense oligomer of the present invention is administered sequentially or simultaneously with another alternative therapy, the other alternative therapy involving the treatment, prevention or improvement of the effects of a disease associated with PTP1B.

[0231] If the disease is associated with insulin resistance, type 2 diabetes mellitus (T2DM), leptin resistance, or obesity, alternative therapies may be selected from a list that includes the following: insulin and insulin mimics; preparations that increase insulin release (dextrin mimics, such as pramlintide; sodium-glucose transporter 2 inhibitors, such as canagliflozin; incretin mimics (GLP-1 agonists), such as exenatide or liraglutide; dipeptidyl peptidase 4 inhibitors, such as saxagliptin, sitagliptin, or linagliptin; sulfonylureas, such as glyburide, glipizide, glimepiride, chlorpropamide, tolazamide, gliquidone, etc. Glibenclamide, gliclazide, acetohexamide, or tolbutamide; meglitinides, such as nateglinide or repaglinide; agents that reduce the absorption of sugar from the intestines (e.g., acarbose, voglibose, and miglitol); agents that prevent the kidneys from reabsorbing filtered glucose (e.g., dapagliflozin and canagliflozin); agents that make the body more sensitive to insulin (e.g., metformin, ciglitazone, troglitazone, rosiglitazone, and pioglitazone); dietary adjustments combined with regular exercise; and surgery to accelerate weight loss.

[0232] If the disease is associated with cancer, alternative therapies can be selected from a list that includes the following: chemotherapy, radiation therapy, surgical resection of solid tumors, and immunotherapy.

[0233] Overview

[0234] Throughout this specification, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” shall be understood to imply inclusion of the stated integers or groups of integers, but not to exclude any other integers or groups of integers.

[0235] Throughout this specification, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” shall be understood to imply inclusion of the stated integers or groups of integers, but not to exclude any other integers or groups of integers.

[0236] Other definitions of the selected terms used herein can be found in the detailed description of the invention and throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0237] Those skilled in the art will understand that, in addition to those specifically described, the invention described herein is susceptible to variations and modifications. This invention includes all such variations and modifications. The invention also includes all steps, features, formulations, and compounds individually or collectively mentioned or indicated in this specification, as well as any two or more of any and all combinations or steps or features.

[0238] Every reference, bibliography, patent application, or patent cited in this article is explicitly incorporated in its entirety by reference, meaning that the reader should read them and consider them part of this article. References, bibliography, patent applications, or patents cited in this article are not repeated here solely for the sake of brevity.

[0239] Any manufacturer's instructions, descriptions, product specifications, and product manuals, used for any product mentioned herein or in any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of this invention.

[0240] The invention described herein may include one or more ranges of values ​​(e.g., concentration). A range of values ​​will be understood to include all values ​​within that range, including the value that defines the range and values ​​adjacent to the range that result in the same or substantially the same result as the value immediately adjacent to the boundary of the defined range.

[0241] The following examples are to be interpreted in any way as illustrative only and not as limiting of the remainder of this disclosure. These examples are given for purposes of illustrative purposes only and should not be construed as limiting the scope of the broad overview, disclosure, or description of the invention as described above. Without further detail, it is believed that those skilled in the art will be able to utilize the invention to its fullest extent using the foregoing description. In the foregoing and the following examples, all temperatures are listed in degrees Celsius without correction; and unless otherwise indicated, all parts and percentages are by weight. Example

[0242] Further features of the invention are described more fully in the following description of several non-limiting embodiments of the invention. This description is given for illustrative purposes only and should not be construed as a broad overview, disclosure, or limitation of the invention as described above.

[0243] Example 1

[0244] Forty-one ASOs (AOs) targeting human PTPN1 or mouse Ptpn1 transcripts were designed and synthesized, as shown in Table 1. All these AOs were transfected into Huh-7 and / or HepG2 cell lines (human hepatocellular carcinoma cell lines); and / or IHH cell line (a normal human hepatocyte cell line); and / or AML-12 cell line (a normal mouse hepatocyte cell line). Results showed that AOs 1-4, 10-15, 18, 19, 23-25, 27, 29, and 31-41 could induce exon skipping (…). Figures 2 to 5 8, 10 to 15, 17 to 19).

[0245] AOs (AO1, AO31-36) targeting exon 2 of human PTPN1 were transfected into liver-related cell lines such as Huh-7. Figure 2 , 5 , 13B), HepG2 ( Figure 8 , 10 11, 13A, 14A), IHH ( Figure 12 , 13 C, 14B, 15), and even mouse AML-12 ( Figure 18 , 19 When transfected into HepG2, it exhibited excellent exon 2 skipping effect. To further evaluate the exon 2 skipping effect of AO in mice, AO (AO 37-41) targeting mouse Ptpn1 exon 2 was transfected into HepG2 (… Figure 17 ) and AML-12 cells ( Figure 18 , 19In addition, AO 1 and 32-36 were found to be highly effective in inducing exon 2 skipping in human PTPN1. Furthermore, AO 33 (PTPN1 1E2A (+5+29))(Diabexa-2) showed the best exon 2 skipping efficiency. Figure 12-14 In addition, its mouse form or version AO 41 (Ptpn1 1E2A (+5+29)) compared to other targeted mouse Ptpn1 exon 2 AO (AO 37-40) also showed improvement in the mouse AML-12 cell line ( Figure 18 , 19 In, and even in the human HepG2 cell line Figure 17 The highest percentage of exon 2 skipping was induced in this process.

[0246] General methods

[0247] Design and synthesis of antisense oligonucleotides

[0248] At the 1 μl oligol scale, through standard phosphoridamide chemistry processes, in ABI 2'-O-methyl (2'OMe)AO (Table 1) was designed and synthesized in-house using an 8909 nucleic acid synthesis system (Applied Biosystems) or a GE AKTA Oligopilot 10 synthesizer (GE Healthcare LifeScience). The synthesized AO was deprotected and cleaved from the solid support by treatment with ammonium hydroxide (NH4OH) at 55 °C for 8 hours. The crude AO was then desalted using an Illustra NAP-10 column (GE Healthcare). The purified oligonucleotides were then validated by polyacrylamide gel electrophoresis.

[0249] Cell culture and ASO transfection into cells

[0250] Human hepatocellular carcinoma cell line Huh-7 was obtained from the American Type Culture Collection (ATCC); another human hepatocellular carcinoma cell line, HepG2, was obtained from the European Collection of Authenticated Cell Cultures (ECACC). Huh-7 cells were cultured in 10% fetal bovine serum (FBS) Dalberg's modified Eagle's medium (DMEM) (Thermo Fisher Scientific), and HepG2 cells were cultured in 10% FBS Eagle's minimum essential medium (ATCC). IHH and AML-12 cell lines were cultured in 10% FBS, 1% ITS (insulin-transferrin-sodium) liquid medium supplement (Thermo Fisher Scientific), and 40 ng / mL dexamethasone (Sigma). All hepatocyte lines were cultured at 55°C under humidification with 5% CO2. Cells were cultured to 70-90% confluence and then injected at 5.0 × 10⁻⁶ cells / mL 24 hours before transfection. 4 Seeds were generated at a density of (cells / mL) into 24-well plates (ThermoFisher Scientific). The next day, transfection was performed according to the original or modified manufacturer's protocol (different modified transfection protocols are based on the manufacturer's instructions and are shown in...). Figure 6 AO cells were transfected at a concentration of 400 nM using RNAiMAX reagent for selection purposes. Cells were collected 24 hours after transfection for RNA extraction.

[0251] RNA extraction and RT-PCR

[0252] According to the manufacturer's instructions, use Direct-zol TM RNA was extracted from transfected cells using RNA MinPrep Plus and TRI reagent (ZymoResearch). III. A one-step RT-PCR kit (ThermoFisher Scientific) and human PTPN1 primer pairs (PTP1B_Ex1F: 5'-GTG ATG CGT AGT TCC GGC TG-3'; PTP1B_Ex6R: 5'-CAG GGA CTC CAA AGT CAG GC-3') or mouse Ptpn1 primer pairs (Ptpn1_B_Ex1F: 5'-AGA TCG ACA AGG CTG GGA AC-3'; Ptpn1_B_Ex6R: 5'-TGA GCC TGA CTC TCG GACTT-3') were used to amplify the human PTPN1 exon 2 skipping product (product size: 639bp) and non-skipping product (product size: 730bp), as well as the mouse Ptpn1 exon 2 skipping product (product size: 493bp) and non-skipping product (product size: 584bp). In summary, the conditions were: 55°C for 30 minutes; 94°C for 2 minutes, followed by 33 cycles of 94°C for 30 seconds, 60°C for 1 minute, and 68°C for 2 minutes. The PCR products were then separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized using the Fusion Fx gel visualization system. Density quantification was performed using ImageJ software.

[0253] sequencing

[0254] Follow the Bandstab technique as described in Anthony and James's guide (1992). Then, use AmpliTaq. The DNA polymerase kit (Thermo Fisher Scientific) was used to amplify the bandstab samples using the same primer set mentioned above. In short, the conditions were: 94°C for 6 minutes; followed by 32 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes. The PCR products were confirmed by 2% agarose gel electrophoresis and then sent to the AGRF (Australian Genome Research Facility) for Sanger sequencing using the forward and reverse primers mentioned above.

[0255] Example 2

[0256] The sequences of PTPN1 1E2A(+1+25)(AO1) and PTPN1 1E2A(+3+27)(AO 32) are similar to one of the AOs patented by Ionis Pharmaceuticals: PTPN1 1E2A(+1+20)(ISIS 107773). AO 1 and AO 32 are chemically treated with 2'-OMePS, while ISIS 107773 is chemically treated with a 5-10-5 MOE (2'-O-methoxyethyl) gapmer. The 2'OMePS forms of AO 1, 32-36, the 2'OMePS form of ISIS 107773, and the 5-10-5 MOE gapmer form of ISIS 107773 were compared in terms of their ability to induce PTPN1 exon 2 skipping. Figure 8 , 10 (Table 2). The data indicate that AO 33 (Diabexa-2) is the best performing AO in inducing PTPN1 exon 2 skipping and / or full-length transcript knockdown. Moreover, AO 33-36 has less than 70% sequence similarity to ISIS 107773 (Table 3).

[0257] Table 2. Comparison of PTPN1 exon 2 skipping efficacy and full-length transcript knockdown efficacy among AO 1, 32-36 and ISIS 107773.

[0258]

[0259]

[0260] Table 3. Sequence comparisons between AO 1, 32-36 and ISIS 107773

[0261]

[0262] The ability of AOs (AO 1, AO 31-36) targeting PTPN1 exon 2 to induce exon 2 skipping has been confirmed. For example, the ability of AO 1 to induce exon 2 skipping has been confirmed by Sanger sequencing. Figure 9 ).

[0263] Example 3

[0264] Different concentrations (400, 200, 100, 50, 25, 12.5 nanomolars) of AO targeting human PTPN1 or mouse Ptpn1 exon 2 were transfected into different types of liver-associated cells, showing a dose-dependent effect. For example, AO 1 induced effective exon 2 skipping in HepG2 cells in a dose-dependent manner. Figure 11AO 33 (Diabexa-2) induces effective exon 2 skipping in HepG2 and IHH cells in a dose-dependent manner. Figure 14 AO 38 (the mouse version of AO 32) and AO41 (the mouse version of AO 33) induced effective exon 2 skipping in mouse AML-12 cells in a dose-dependent manner. Figure 19 (C, D). All of the above results confirm that AOs targeting PTPN1 exon 2 (AO 1, AO 32-36), with the most preferred AO 33 (Diabexa-2) (PTPN1 1E2A(+5+29)) inducing significant PTPN1 transcript exon 2 skipping, and therefore these AOs may potentially induce a reduction in the production of functional PTP1B protein.

[0265] The results clearly demonstrate that AO 33(Diabexa-s)(PTPN1 1E2A(+5+29)) exhibits better human PTPN1 exon 2 skipping and non-skipping product knockdown effects than other AOs targeting exon 2, including AO 1, 32, 34-36, and the 2'-OMePS and 5-10-5MOE gapmer forms of ISIS 107773. Figure 12-14 Furthermore, compared to other AOs targeting Ptpn1 exon 2 (AO 37-40), the mouse form of AO 33 (Diabexa-2), namely AO 41, showed the best mouse Ptpn1 exon 2 skipping effect. Figure 17-19 ).

[0266] Example 4

[0267] The phosphoridamide morpholino oligomer (PMO) form of AO targeting exon 2 was synthesized and evaluated. For example, the PMO form of AO 33 (Diabexa-2) was transfected into IHH cells via nuclear transfection and showed effective PTPN1 exon 2 skipping in a dose-dependent manner. Figure 15 All of the above results confirm that AO 33 (Diabexa-2) induces significant exon 2 skipping in the PTPN1 transcript, and therefore this AO may potentially induce a reduction in the production of functional PTP1B protein.

[0268] Western blotting was performed to assess the effects of the 2' OMePS and PMO forms of PTPN1 1E2A(+5+29) on PTP1B protein inhibition compared to untreated samples. IHH cells were harvested 72 hours post-transfection and stored at -80°C. Frozen transfected IHH cell clusters were thawed and homogenized in SDS lysis buffer containing a protease inhibitor (Sigma) (0.5 M Tris-HCl pH 6.8, 3% SDS (w / v), and 10% glycerol (v / v)). The homogenate was then centrifuged at 14,000 g for 3 minutes, and the supernatant was collected and processed using Pierce chromatography. TM The protein concentration in the supernatant was estimated using the BCA Protein Assay Kit (ThermoFisher Scientific). Proteins were then separated from the sample on a nitrocellulose membrane (Biorad). The membrane was incubated overnight at 4°C with primary antibody PTP1B (1:1000) (Cat. 5311S, CellSignaling Technology) in 5% skim milk in a TBS-T solution. The membrane was then washed with TBS-T at room temperature for 1 hour on a shaker. After washing, the membrane was incubated with secondary antibody rabbit HRP (1:10000) (Cat. 31460, ThermoFisher Scientific) on a shaker at room temperature for 1 hour, followed by TBS-T-based washing. Protein bands were visualized using a chemiluminescence-based procedure using the Clarity Western ECL Detection Kit, following the manufacturer's instructions (Biorad). Western blot results showed that PTPN1 1E2A(+5+29)(AO 33) treatment significantly reduced the expression level of PTP1B protein. Figure 16 Specifically, 400 nanomolars of the 2' OMePS form of AO 33 (Diabexa-2) induced a 31% reduction or inhibition of PTP1B protein, while 7.5 μM and 15 μM of the PMO form of AO 33 (Diabexa-2) induced 20% and 50% reductions or inhibition of PTP1B protein, respectively. Figure 16 ).

[0269] AO 1 and 31-36 exhibited superior exon 2 skipping in human PTPN1, leading to the induction of a premature stop codon in exon 3 and a significant reduction in the expression level of the functional PTPN1 gene product. Furthermore, Western blot results have demonstrated that AO 33(Diabexa-2):PTPN1 1E2A(+5+29) treatment significantly reduced the expression level of PTP1B protein.

[0270] Example 5

[0271] Analysis of PTP1B expression in various cancer cells, including breast cancer (MCF-7, MDA), mesothelioma (JU77, One58), glioblastoma (U87, U251), neuroblastoma (SH-SY5Y), medulloblastoma (DAOY), and hepatocellular carcinoma (HepG2), revealed high expression of PTP1B. Figure 20 The following primer pairs were used to obtain a 730bp product (PTP1B_Ex1F: 5'-GTGATG CGT AGT TCC GGC TG-3'; PTP1B_Ex6R: 5'-CAG GGA CTC CAA AGT CAG GC-3'). In short, the conditions were: 55°C for 30 min; 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 1 min, and 68°C for 2 min. The PCR products were then separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized using the Fusion FX gel documentation system. SEQUENCE LISTING <110> Murdoch University <120> Antisense therapy for PTP1B-related conditions <130> P21H61627A <150> AU2018903950 <151> 2018-10-18 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> RNA <213> Homo sapiens <400> 1 agucacuggc uucaugucgg auauc 25 <210> 2 <211> 25 <212> RNA <213> Homo sapiens <400> 2 uagucauuau cuucuugaug uaguu 25 <210> 3 <211> 25 <212> RNA <213> Homo sapiens <400> 3 uguaacuccu uugggcuucu uccau 25 <210> 4 <211> 25 <212> RNA <213> Homo sapiens <400> 4 augacuugau aucuucagag aucaa 25 <210> 5 <211> 25 <212> RNA <213> Homo sapiens <400> 5 gggaaagcuc cuuccacuga uccug 25 <210> 6 <211> 25 <212> RNA <213> Homo sapiens <400> 6 cauugugugg cuccaggauu cguuu 25 <210> 7 <211> 25 <212> RNA <213> Homo sapiens <400> 7 ucucuuccuu caccacugg ugauu 25 <210> 8 <211> 25 <212> RNA <213> Homo sapiens <400> 8 cgaugccgua gggugcggca uuuaa 25 <210> 9 <211> 25 <212> RNA <213> Homo sapiens <400> 9 gcuccggggc gcuccgcac cuggu 25 <210> 10 <211> 25 <212> RNA <213> Homo sapiens <400> 10 aauaccuaca aaaaagaaua aagac 25 <210> 11 <211> 25 <212> RNA <213> Homo sapiens <400> 11 ccggccacgu gguaacuuac aggga 25 <210> 12 <211> 25 <212> RNA <213> Homo sapiens <400> 12 gucaacugaa agacaaacca gaacu 25 <210> 13 <211> 25 <212> RNA <213> Homo sapiens <400> 13 aauucagaca aucugcuuac cuggg 25 <210> 14 <211> 25 <212> RNA <213> Homo sapiens <400> 14 gggcccugca aagacacaau aacac 25 <210> 15 <211> 25 <212> RNA <213> Homo sapiens <400> 15 caaaugaagc cgagacuuac cgaac 25 <210> 16 <211> 25 <212> RNA <213> Homo sapiens <400> 16 uuuaacugg gaaacaaaua auagu 25 <210> 17 <211> 25 <212> RNA <213> Homo sapiens <400> 17 gaagugugug cuauacucac uguaa 25 <210> 18 <211> 25 <212> RNA <213> Homo sapiens <400> 18 ugggucugaa agagaaaaau acuca 25 <210> 19 <211> 25 <212> RNA <213> Homo sapiens <400> 19 acccgcgagg gccccuuac cagca 25 <210> 20 <211> 25 <212> RNA <213> Homo sapiens <400> 20 uccaucugaa agccagagag gagau 25 <210> 21 <211> 25 <212> RNA <213> Homo sapiens <400> 21 caaacaaagg caaugcugac cugca 25 <210> 22 <211> 25 <212> RNA <213> Homo sapiens <400> 22 ugauccuuga aagagcagca agagg 25 <210> 23 <211> 25 <212> RNA <213> Homo sapiens <400> 23 cugggaccca aucauauuac cuuuuc 25 <210> 24 <211> 25 <212> RNA <213> Homo sapiens <400> 24 ucaugcugag gaaucagagg gcaga 25 <210> 25 <211> 25 <212> RNA <213> Homo sapiens <400> 25 gucugucagu ggaaacauac ccugu 25 <210> 26 <211> 25 <212> RNA <213> Homo sapiens <400> 26 aggaacugga augaaaccaa acagu 25 <210> 27 <211> 25 <212> RNA <213> Homo sapiens <400> 27 cgacuucuaa cuucaguguc uugac 25 <210> 28 <211> 25 <212> RNA <213> Homo sapiens <400> 28 ugacggcucc ccuuuggcug gggag 25 <210> 29 <211> 25 <212> RNA <213> Homo sapiens <400> 29 ucagugcaug guccucgucc uucuc 25 <210> 30 <211> 25 <212> RNA <213> Homo sapiens <400> 30 agagguaagc gccggccgug aggac 25 <210> 31 <211> 23 <212> RNA <213> Homo sapiens <400> 31 ucacuggcuu caugucggau auc 23 <210> 32 <211> 25 <212> RNA <213> Homo sapiens <400> 32 gaagucacug gcuucauguc ggaua 25 <210> 33 <211> 25 <212> RNA <213> Homo sapiens <400> 33 gggaagucac uggcuucaug ucgga 25 <210> 34 <211> 25 <212> RNA <213> Homo sapiens <400> 34 augggaaguc acuggcuuca ugucg 25 <210> 35 <211> 25 <212> RNA <213> Homo sapiens <400> 35 acaugggaag ucacuggcuu caugu 25 <210> 36 <211> 25 <212> RNA <213> Homo sapiens <400> 36 cuacauggga agucacuggc uucau 25 <210> 37 <211> 25 <212> RNA <213> Homo sapiens <400> 37 agucgcuggc uucaugucga auguc 25 <210> 38 <211> 25 <212> RNA <213> Homo sapiens <400> 38 gaagucgcug gcuucauguc gaaug 25 <210> 39 <211> 25 <212> RNA <213> Homo sapiens <400> 39 cuggcuucau gucgaauguc cuaca 25 <210> 40 <211> 25 <212> RNA <213> Homo sapiens <400> 40 ucgcuggcuu caugucgaau auccu 25 <210> 41 <211> 25 <212> RNA <213> Homo sapiens <400> 41 gggaagucgc uggcuucaug ucgaa 25

Claims

1. An isolated or purified antisense oligomer that targets a nucleic acid molecule encoding PTPN1 precursor mRNA, wherein the nucleobase sequence of the antisense oligomer is selected from the list consisting of SEQ ID NO: 1 and 32-36, wherein SEQ ID NO: 1 and 32-36 have a modified backbone structure, wherein the antisense oligomer induces alternative splicing of PTPN1 precursor mRNA by exon 2 skipping, and wherein the antisense oligomer inhibits the expression of PTP1B.

2. The antisense oligomer according to claim 1, wherein: a) The antisense oligomer contains one or more nucleotide positions that have undergone selective chemical processes or modifications selected from a list including: (i) modified sugar moieties; (ii) resistance to RNase H; (iii) oligomerization mimicry processes; b) The antisense oligomer is further modified by: (i) chemically conjugating a portion; and / or (ii) labeling with a cell-penetrating peptide; and / or c) If uracil (U) is present in the antisense oligomer, then the uracil (U) in the antisense oligomer is replaced by thymine (T).

3. The antisense oligomer according to claim 1 or 2, wherein: a) Phosphoridamide morpholine oligomer (PMO); b) 2'-O-methoxyethyl RNA oligomer (2'-O-MOE); or c) 2'-O-methylRNA (2'-OMe) oligomer.

4. The antisense oligomer according to any one of claims 1-3, wherein it is SEQ ID NO:

33.

5. An expression vector comprising the antisense oligomer of any one of claims 1-4.

6. A pharmaceutical composition comprising: One or more antisense oligomers according to any one of claims 1 to 4; and One or more drug-acceptable carriers and / or diluents.

7. Use of the purified and isolated antisense oligomer according to any one of claims 1 to 4 for the preparation of a medicament for the treatment, prevention, or improvement of the effects of PTP1B-associated disease in a subject. The diseases associated with PTP1B are selected from type 2 diabetes, obesity, and solid tumors, with the solid tumors selected from liver cancer, ovarian cancer, gastric cancer, prostate cancer, breast cancer, mesothelioma, glioblastoma, neuroblastoma, and medulloblastoma.

8. A kit comprising at least an antisense oligomer according to any one of claims 1 to 4 packaged in a suitable container, and instructions for use.

Images