Thiomorpholino antisense oligonucleotides for treating PTP1B-related diseases

Abstract

The present invention relates to antisense oligonucleotides (ASOs) used to treat, prevent, or mitigate the progression of conditions such as type 2 diabetes mellitus (T2DM) and insulin resistance, leptin resistance and obesity, Rett syndrome, and cancer. Specifically, thiomorpholino-containing ASOs target the protein tyrosine phosphatase non-receptor type 1 (PTPN1) gene transcript during RNA processing and induce exon skipping (including exon 2), thereby inhibiting the expression of protein tyrosine phosphatase-1B (PTP1B) protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 425,161, filed November 14, 2022, the specification, claims and drawings of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application includes the contents of the electronic sequence listing (90245-00881-Sequence-Listing.xml, size: 37,163 bytes, created on November 2, 2023), the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to antisense oligonucleotides (ASOs) containing thiomorpholino (TMO, Figure 21) nucleotides for treating, preventing, or mitigating the progression of conditions such as type 2 diabetes mellitus (T2DM) and insulin resistance, leptin resistance and obesity, Rett syndrome, and cancer. In a preferred embodiment, the thiomorpholino-containing ASO targets the protein tyrosine phosphatase non-receptor type 1 (PTPN1) gene transcript during RNA processing and induces exon skipping (including exon 2), thereby inhibiting expression of the protein tyrosine phosphatase-1B (PTP1B) protein. The present invention further provides methods for treating, preventing, or mitigating the effects of insulin resistance and leptin resistance by administering antisense oligomers (ASOs) and therapeutic compositions containing the antisense oligomers to the PTPN1 gene. [Background technology]

[0004] The discussion of background art is intended solely to facilitate understanding of the present invention. The discussion is not an admission or certification that any of the referenced materials are or were part of the common general knowledge at the priority date of the application. Currently, the three main chemical approaches widely used in splice switching applications are phosphorodiamidate morpholino oligomers (PMOs), 2'-O-methyl (2'-OMe) and 2'-O-methoxyethyl (2'-MOE) (Figure 22). However, these chemical approaches have significant limitations, such as toxicity in the case of 2'-OMe and 2'-MOE [1, 2]. Meanwhile, PMOs, despite their excellent safety profile, are difficult to synthesize on a scale sufficient for use as therapeutic agents. Furthermore, PMOs are known to be rapidly excreted after in vivo administration, thereby requiring high doses and potentially contributing to treatment costs. Another limitation of PMOs is their inability to form complexes with commercially available transfection reagents, preventing rapid evaluation of PMO drugs in in vitro systems.

[0005] Protein tyrosine phosphatase-1B (PTP1B, Figure 23) is a member of the protein tyrosine phosphatase (PTP) superfamily [3]. It was the first PTP molecule discovered and is therefore the most extensively studied to date. The 435 amino acid (50 kDa) protein is primarily localized in the endoplasmic reticulum (ER) in the cytoplasm. PTP1B functions by dephosphorylating various growth factor receptors and tyrosine kinases involved in numerous metabolic pathways, such as insulin, leptin, and brain-derived neurotrophic factor (BDNF) signaling pathways, as well as oncogenic pathways, such as the proto-oncogene tyrosine-protein kinase Src, pituitary homeobox 1 (PITX1), and Ras GTPase [3, 4]. Therefore, PTP1B has clinical implications in the treatment of type 2 diabetes, obesity, Rett syndrome, and cancer.

[0006] In the insulin signaling pathway, PTP1B acts as one of the major negative regulators of the pathway [3, 4]. In normal individuals, glucose homeostasis (glucose uptake) occurs when insulin binds to the insulin receptor, followed by recruitment of insulin receptor substrates and activation of the Pl3K / Akt signaling pathway. This pathway promotes glucose transporter type 4 (GLUT4) translocation from the cytoplasm to the membrane, facilitating glucose uptake [4] (Figure 24A). In type 2 diabetes patients, PTP1B dephosphorylates the insulin receptor and insulin receptor substrates, thus disrupting the Pl3K / Akt signaling pathway. This prevents GLUT4 translocation to the plasma membrane and prevents glucose uptake into the cell. Cells may require significantly more insulin to promote glucose uptake, again resulting in insulin insensitivity and insulin resistance (Figure 24B). Inhibiting PTP1B expression restores the P13K / Akt signaling pathway and therefore GLUT4 translocation, resulting in normal glucose uptake into cells (FIG. 24C).

[0007] In the leptin signaling pathway, leptin binds to the leptin receptor and activates the JAK2 / STAT3 signaling pathway, resulting in energy homeostasis [3, 4]. This pathway regulates energy expenditure and food intake, helping to control body weight (Figure 25A). In obese individuals, PTP1B dephosphorylates the leptin receptor and JAK2 substrates, thus interfering with the JAK2 / STAT3 signaling pathway. This leads to overnutrition, increased food intake, and ultimately obesity (Figure 25B). Inhibiting PTP1B expression restores the JAK2 / STAT3 signaling pathway, resulting in normal regulation of energy expenditure, food intake, and body weight control (Figure 25C).

[0008] Previous studies have also demonstrated that PTP1B inhibition has a positive effect in animal models of Rett syndrome [5, 6]. Rett syndrome (RTT) is a neurological disorder that affects approximately 1 in 10,000 live births [6]. This condition is caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene, which leads to impaired function of the MECP2 protein. In normal individuals, MECP2 functions by targeting the neurotrophic factor brain-derived neurotrophic factor (BDNF) and regulating the BDNF / TRKB (tropomyosin receptor kinase B) pathway to maintain cell survival, cell differentiation, and synaptic activity [5, 6]. Furthermore, MECP2 has been found to directly bind to the promoter region of the PTPN1 gene, suppressing the gene's production of PTP1B protein [5, 6] (Figure 26A). In patients with Rett syndrome, mutations in the MECP2 gene lead to impaired function of the MECP2 protein. This results in overexpression of PTP1B levels in cells, followed by dephosphorylation of the TRKB receptor and suppression of the BDNF / TRKB pathway (Figure 26B). By inhibiting PTP1B expression, the function of the BDNF / TRKB pathway is restored, although MECP2 protein function remains impaired (Figure 26C). This has been shown to improve symptoms and survival in animal models of Rett syndrome [6].

[0009] Apart from its function in metabolic pathways, PTP1B has also been found to play an important role in various tumorigenesis pathways. In breast cancer, PTP1B inhibits the p62Dok and p120RasGap complex, thereby enhancing Ras-mediated cell proliferation, growth, and migration [3]. Similarly, PTP1B-mediated dephosphorylation of Src leads to the subsequent activation of Ras-mediated pathways. In parallel, the interaction between Grb2 and the HER2 / HER3 receptor also leads to the activation of the proto-oncoprotein Ras. These pathways result in tumor cell proliferation, growth, and migration (Figure 27A). Inhibiting PTP1B expression restores the inhibitory effects of p62Dok / p120RasGap and Src on the Ras proto-oncoprotein, thereby resulting in tumor suppression (Figure 27B).

[0010] In pancreatic cancer, PTP1B dephosphorylates multiple proteins, including the p62Dok / p120RasGap complex and the proto-oncogene Src, thereby enhancing Ras-mediated cell proliferation [7]. Furthermore, PTP1B has been found to have an indirect inhibitory effect on the P13K / AKT pathway, leading to tumor cell survival. In another context, PTP1B-mediated dephosphorylation also acts on the PKM2 protein to enhance tumor cell growth. In combination, PTP1B has a positive effect on the proliferation, survival, and growth of pancreatic cancer cells (Figure 28A) [7]. Inhibiting PTP1B expression restores the inhibitory effects of the p62Dok / p120RasGap complex and the proto-oncogene Src on Ras proteins. Furthermore, the P13K / AKT and PKM2 pathways also restore their normal function. In conclusion, PTP1B inhibition results in a combined tumor-suppressive effect (Figure 28B).

[0011] In liver cancer, PTP1B has been proposed to dephosphorylate PITX1 protein, disrupting the inhibitory effect of PITX1 on the GTPase-activating protein p120RasGap [8]. This leads to tumorigenesis in the liver (Figure 29A). Inhibiting PTP1B expression restores the PITX1 inhibitory function on p120RasGap, resulting in tumor suppression (Figure 29B).

[0012] The present invention aims to provide compositions and methods for reducing the effects of insulin resistance, T2DM, leptin resistance, obesity, Rett syndrome and cancer, or for providing consumers with useful or commercial options.As described below, in one embodiment, the inventors have developed thiomorpholino (TMO) antisense oligonucleotides (AO or ASO) to induce exon 2 skipping in PTPN1 pre-mRNA transcripts, thereby eliminating the expression of PTP1B protein.For this purpose, various exon skipping ASOs have been designed, synthesized and evaluated in in vitro model systems. Summary of the Invention

[0013] The present invention is based on the surprising discovery that the use of isolated or purified AO to alter the pre-mRNA splicing production of PTPN1 to increase the production of truncated, nonsense or prematurely terminated proteins, such as proteins with premature stop codons, can result in reduced production of functional PTP1B protein.

[0014] Generally, according to one aspect of the present invention, there is provided an isolated or purified AO for altering pre-mRNA splicing in the protein tyrosine phosphatase-1B (PTP1B) protein, or a portion thereof, encoded by the PTPN1 gene transcript. Preferably, there is provided an isolated or purified antisense oligomer for inducing splice modulation, particularly exon skipping, leading to a premature stop codon that results in reduced production of the full-length PTPN1 gene transcript, or a portion thereof.

[0015] Preferably, the antisense oligomer is a morpholino oligomer having 3'-phosphorothioamidate internucleotide linkages, which may be generally referred to herein as a thiomorpholino oligonucleotide (TMO) as described herein.

[0016] Preferably, the antisense oligomer is selected from the group comprising the sequences set forth in Table 1 and Table 4. Preferably, the antisense oligomer is selected from the table comprising SEQ ID NOs: 42 to 57, or 69 to 73 or 75. Preferably, the antisense oligomer used in the present invention is selected from the table comprising SEQ ID NOs: 42 to 57 and / or 69 to 73 or 75. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 42, 46, 50, 52.

[0017] According to a still further aspect, the present invention extends to cDNA or cloned copies of the antisense oligomer sequences of the invention, and to vectors containing the antisense oligomer sequences of the invention. The present invention further extends to cells containing such sequences and / or vectors.

[0018] Also provided is a method for manipulating splicing factor binding in a PTPN1 gene transcript, the method comprising: This includes providing one or more of the antisense oligomers described herein and allowing the oligomer(s) to bind to the target nucleic acid site.

[0019] Also provided are pharmaceutical, prophylactic or therapeutic compositions for treating, preventing or ameliorating the effects of a disease associated with a PTP1B protein in a subject, the compositions comprising: one or more antisense oligomers described herein; one or more pharmaceutically acceptable carriers and / or diluents; Includes.

[0020] Preferably, the disease states associated with PTP1B protein are insulin resistance, type 2 diabetes mellitus (T2DM), leptin resistance, obesity, Rett syndrome and cancer.

[0021] The subject having a disease associated with the PTP1B protein may be a mammal, including a human.

[0022] Also provided is a method for treating, preventing, or ameliorating the effects of a disease associated with a PTP1B protein, the method comprising: The method includes administering to a subject an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers described herein.

[0023] Also provided is the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with the PTP1B protein.

[0024] Kits for treating, preventing, or alleviating the effects of a disease associated with a subject's PTP1B protein are also provided, which contain at least the antisense oligomers and combinations or cocktails thereof described herein packaged in a suitable container, along with instructions for use thereof.

[0025] Further aspects of the present invention will now be described with reference to the accompanying non-limiting examples and figures.

[0026] Further features of the present invention will be more fully described in the following description of several non-limiting embodiments thereof. This description is included for purposes of illustrating the present invention only. It should not be understood as a limitation on the broad summary, disclosure, or description of the present invention set forth above. The following description refers to the accompanying drawings. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is an exon map of the human and mouse Ptpn1-201 transcripts. [Figure 2] Northern blot showing the transfection efficiency of AO1-8 in Huh-7 cell line at 400 nanomolar concentration. S: scrambled sequence, UT: untreated, NC: negative control, Imax: RNAiMAX, pro: Metafectene® PRO, si: Metafectene® SI+. [Figure 3] Northern blot showing transfection efficiency of AO9-16 using RNAiMAX reagent in Huh-7 cell line at 400 nanomolar concentration. S: scrambled sequence, UT: untreated, NC: negative control. [Figure 4] Northern blot showing the transfection efficiency of AO17-30 using L3K reagent in Huh-7 cell line at 400 nanomolar concentration. S: scrambled sequence, UT: untreated, NC: negative control. [Figure 5] Northern blot showing a dose-response study of AO1 and AO4 at concentrations including 400, 200, 100, 50, 25, and 12.5 nmol in Huh-7 cell line. S: scrambled sequence, UT: untreated, NC: negative control. [Figure 6] This is a schematic diagram of the modified transfection protocol used for HepG2 transfection experiments based on the manufacturer's instructions for RNAiMAX and Lipofectamine 3000 (L3K). The 2'OMePS form from AO1 was used in this experiment. The stock concentration of AO1 is 0.181171 millimolar (mM). Opti is an abbreviation for Opti-MEM™ I reduced serum medium. [Figure 7]Northern blot showing that different transfection reagents (RNAiMAX and L3K) and different transfection protocols (RNAiMAX: 1.1–1.7, L3K: 2.1 and 2.2) were tested in the HepG2 cell line. The results showed that protocol 1.3 (the reverse transfection protocol of RNAiMAX) was the best protocol for 2′OMePS antisense oligonucleotide transfection into HepG2. The 2′OMePS form of AO1 was used in this experiment. [Figure 8] Northern blot showing a comparison of exon-2 skipping efficiency between PTPN1 1E2A(+1+25)(AO1), the 2′-OMePS form of ISIS 107773, PTPN1 1E2A(+1+23)(AO31), and PTPN1 1E2A(+3+27)(AO32) at a concentration of 400 nanomolar in HepG2. [Figure 9] AO1, Sanger sequencing results confirming that PTPN1 1E2A(+1+25) induces exon-2 skipping during the transcription process of gene PTPN1. [Figure 10] Northern blot showing a comparison of exon-2 skipping and non-skipping product knockdown efficiencies between PTPN1 1E2A(+1+25)(AO1), PTPN1 1E2A(+3+27)(AO32), the 2′-OMePS form of ISIS 107773, and ISIS 107773 (5-10-5 MOE gapmer) at 400 nanomolar concentrations in triplicate in HepG2. [Figure 11] Northern blot showing a dose response study of PTPN1 1E2A(+1+25)(AO1) at concentrations including 400, 200, 100, 50, 25, 12.5, 6.3, and 3.1 nanomolar in HepG2. [Figure 12] Northern blot showing transfection efficiency of AO1, 32-36 using RNAiMAX reagent in the IHH cell line at 400 nanomolar concentration. [Figure 13]A–C are Northern blots showing a comparison of exon-2 skipping and non-skipping product knockdown efficiencies between AO1, AO32-36, the 2′-OMePS form of ISIS 107773, and ISIS 107773 (5-10-5 MOE gapmer) at 400 nanomolar concentrations using RNAiMAX reagent in the HepG2 cell line. [Figure 14] Northern blot showing a dose response study of the 2'OMePS form of PTPN1 1E2A(+5+29)(AO33)(Diabexa-2) in HepG2 cells. Concentrations include 400, 200, 100, 50, 25, and 12.5 nanomolar. Cells were transfected using RNAiMAX. [Figure 15] Northern blot showing a dose response study of the PMO form of PTPN1 1E2A (+5+29)(AO33)(Diabexa-2) in IHH cells. Concentrations include 30, 15, and 7.5 micromolar. Cells were transfected by nucleofection. [Figure 16] (A-B) Western blots showing the reduction of PTP1B protein production induced by the 20MePS form of AO33 (Diabexa-2) (400 nanomolar) and the PMO form of AO33 (Diabexa-2) (15, 7.5 micromolar) in IHH cells. Cells were harvested 72 hours after transfection with AO. [Figure 17] Northern blot showing the transfection efficiency of AO37-41 (AO targeting mouse Ptpn1 exon-2) using RNAiMAX reagent in HepG2 cell line at 400 nanomolar concentration. [Figure 18] Northern blot showing the transfection efficiency of AO37-41 (AO targeting mouse Ptpn1 exon-2) and AO1, 32, 33 (AO targeting human PTPN1 exon-2) using RNAiMAX or L3K reagents in the mouse AML-12 cell line at an AO concentration of 400 nanomolar. [Figure 19](A-D) Northern blots showing the transfection efficiency of AO37, 38, and 41 (AO targeting mouse Ptpn1 exon-2) and AO1, 32, and 33 (AO targeting human PTPN1 exon-2) in the mouse AML-12 cell line using RNAiMAX. A: AO37 is the mouse version of AO1 (with three mismatches), AO38 is the mouse version of AO32 (with three mismatches), and AO41 is the mouse version of AO33 (with two mismatches). B: Transfection efficiency of AO37, 38, 41, 1, 32, and 33 at 400 nanomolar concentration is shown. C: Dose dependence of AO38 is shown. D: Dose dependence of AO41 is shown. [Figure 20] Figure 1 shows the expression of PTPN1 in cancer cells. The annealing temperatures for the RT-PCR reaction were 57.8°C, 60°C, and 62°C. The PCR cycle was 30 cycles. [Figure 21] A structural representation of a TMO monomer on a phosphorothioamidate backbone is shown. [Figure 22] Structural representations of TMO monomers on a phosphorothioamidate backbone, PMO monomers on an N,N-dimethylaminophosphorodiamidate backbone, and 2'-OMe and 2'-MOE monomers on a phosphorothioate (PS) backbone are shown. [Figure 23] A schematic diagram of the domain structure of the human PTP1B protein (top) and an exon map of the human PTPN1 gene (bottom) are shown. [Figure 24] A to C show PTP1B in the insulin signaling pathway. A shows glucose homeostasis. B shows PTP1B in the insulin signaling pathway and shows insulin resistance. C shows PTP1B inhibition. [Figure 25] A-C show PTP1B in the leptin signaling pathway. A shows energy homeostasis. B shows leptin resistance. C shows PTP1B inhibition. [Figure 26] A to C show PTP1B in Rett syndrome. A: normal individual, B: Rett syndrome patient, C: PTP1B inhibition. [Figure 27] A and B show PTP1B in breast cancer. A: breast cancer patient, B: PTP1B inhibition. [Figure 28] A and B show PTP1B in pancreatic cancer. A: pancreatic cancer patient, B: PTP1B inhibition. [Figure 29] A and B show PTP1B in liver cancer. A: liver cancer individual, B: PTP1B inhibition. [Figure 30] Initial screening of TMO ASOs that induce exon 2 skipping in human PTPN1 pre-mRNA transcripts and densitometry analysis of RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 31] Comparison of 2'-OMe (ASO6) and 2'-MOE (ASO7) with the TMO chemical structure (ASO5) in inducing exon 2 skipping in human PTPN1 pre-mRNA transcripts, and densitometry analysis of the RT-PCR products. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product. UT = untreated sample; Ne = negative. [Figure 32] Evaluation of TMO ASO using microwalking technique against the 5' end of the human PTPN1 pre-mRNA transcript and densitometric analysis of RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 33] Evaluation of TMO ASO using microwalking technique against the 3' end of the human PTPN1 pre-mRNA transcript and densitometric analysis of the RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 34]A-D. (A) Evaluation of TMO ASO (400 nM) targeting human PTPN1 pre-mRNA transcripts at the RNA level (72 h) and (B) densitometric analysis of RT-PCR products. (C) Evaluation of TMO ASO at the protein level (72 h) by Western blot normalized to the GAPDH housekeeping gene and untreated samples. (D) Densitometric analysis of Western blot normalized to the GAPDH housekeeping gene and untreated samples. In (B), gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 35] Evaluation of the 25-mer TMO ASO1 compared to the 18-mer TMOs (ASO5, 9, and 11) and densitometry analysis of the RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product. UT = untreated sample; Ne = negative. [Figure 36] Evaluation of lead TMO ASO in liver cancer cells and densitometry analysis of RT-PCR products. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 37] Evaluation of lead TMO ASO in pancreatic cancer cells and densitometry analysis of RT-PCR products. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 38] Evaluation of lead TMO ASO in triple-negative breast cancer cells and densitometry analysis of RT-PCR products. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 39]Initial screening of TMO ASOs that induce exon 2 skipping in mouse Ptpn1 pre-mRNA transcripts and densitometry analysis of RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 40] A-B: (A) Schematic diagram of human and mouse PTPN1 transcripts in the region of interest in exon 2. (B) Binding positions of mouse ASOs are shown. The green line represents the original test ASO. The blue line represents the newly designed ASO. [Figure 41] Evaluation of the newly designed 2'-MOE ASO inducing exon 2 skipping in mouse Ptpn1 pre-mRNA transcripts and densitometry analysis of the RT-PCR products are shown. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product; UT = untreated sample; Ne = negative. [Figure 42] Evaluation of ASO efficacy by length using 25-mer mouse 2'-MOE ASO23 and 18-mer 2'-MOE ASO21 in inducing exon 2 skipping in mouse Ptpn1 pre-mRNA transcripts and densitometric analysis of RT-PCR products. Triangles above the gel image indicate increasing concentrations of 50, 100, 200, and 400 nM. Gray: exon 2 skipping product; white: full-length product. UT = untreated sample; Ne = negative. [Figure 43] Figures A-B show the evaluation of exon 2 skipping induction in mouse Ptpn1 pre-mRNA transcripts and densitometric analysis of RT-PCR products using a 25-mer mouse TMO ASO24 compared with a 25-mer 2'-MOE ASO23. Triangles over the gel image indicate increasing concentrations (A: 50, 100, 200, and 400 nM; B: 5, 10, 25, and 50 nM). Gray: exon 2 skipping product; white: full-length product. UT = untreated sample; Ne = negative. DETAILED DESCRIPTION OF THE INVENTION

[0028] Antisense oligomers The present 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, T2DM, leptin resistance, obesity, Rett syndrome, and cancer. This alteration of PTP1B expression can be achieved using antisense oligomers (also known as antisense oligonucleotides, ASOs, AOs, and AONs, and these terms are interchangeable).

[0029] Protein tyrosine phosphatase-1B (PTP1B), encoded by the gene PTPN1, is a phosphatase that negatively regulates insulin signaling and is therefore responsible for insulin resistance, one of the underlying causes of T2DM. Apart from blocking insulin signaling, PTP1B also downregulates the leptin signaling pathway, resulting in the reduced energy expenditure and increased fat accumulation associated with obesity, which contributes to insulin resistance and is one of the most important risk factors for T2DM. Because PTP1B simultaneously blocks both insulin and leptin signaling, the present inventors investigated the use of PTPN1 as a target gene for the development of therapeutic agents for T2DM and obesity.

[0030] Without being bound by any theory, the present invention is based on the following understanding. Downregulating PTP1B expression leads to upregulation of insulin signaling, and / or ·Downregulating PTP1B expression leads to upregulation of the leptin signaling pathway.

[0031] The PTP1B protein is also associated with several solid tumor cancers, and knockdown of PTP1B reduces cell proliferation, induces both cell cycle arrest and apoptosis, and reduces cancer cell migration and invasion by reversing the epithelial-mesenchymal transition (EMT) process.

[0032] PTPN1 has 10 exons, including four exons (exons 2, 3, 8, and 9) that contain overlapping splice sites (Figure 1). Two of these exons, exon 2 and exon 3, are located near the 5' end of the transcript. When exon 2 is skipped, a premature stop codon is introduced into exon 3, suggesting that mutant transcripts resulting from exon 2 skipping may not translate into functional PTP1B protein. Alternatively, exon skipping can be used to develop truncated or nonsense PTP1B proteins.

[0033] Preferably, the disease or condition treated or prevented by the antisense oligomer of the present invention is a disease (i) associated with downregulation of insulin signaling in a subject, (ii) associated with downregulation of the leptin signaling pathway in a subject, and / or (iii) associated with dysfunction of the BDNF / TRKB pathway, such as in Rett syndrome, and / or (iv) associated with the growth, migration, and invasion of cancer cells. For example, the disease may be T2DM, obesity, Rett syndrome, or cancer.

[0034] The present invention does not specifically aim 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 cannot be significantly altered (although some changes 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 a translated protein lacking this domain may be unable to catalyze the process of removing phosphate groups from phosphorylated tyrosine residues on proteins. Exons 6 and 7 encode regions containing substrate-binding sites, and removing these exons can generate nonfunctional PTP1B proteins.

[0035] The presence of an internally truncated protein (i.e., a protein lacking the amino acids encoded by one or more exons) is preferred. If the PTP1B protein is knocked out, there may be problems with increasing PTPN1 transcription, as the body attempts to compensate for the reduced total amount of PTP1B protein. In contrast, the presence of an internally truncated protein (preferably lacking one or more characteristics of the complete PTP1B protein) will be sufficient to prevent increased transcription, yet still provide a therapeutic advantage due to the reduced total amount of functional PTP1B protein. Preferably, exon skipping leads to exon 2 skipping, and exon 2 skipping results in the induction of a premature stop codon in exon 3.

[0036] The antisense oligomer-induced exon skipping of the present invention need not completely or even substantially eliminate the function of the PTP1B protein. Preferably, the exon skipping process results in reduced or abolished functionality of the PTP1B protein.

[0037] In contrast to other antisense oligomer-based therapies, the present invention does not induce increased RNA degradation via recruitment of RNase H, which preferentially binds to and degrades RNA double-stranded to the DNA of the PTPN1 gene, and it does not depend on hybridization of the antisense oligomer to PTPN1 genomic DNA or binding of the antisense oligomer to mRNA, but rather regulates the amount of PTP1B protein produced by interfering with normal functions such as replication, transcription, translocation, and translation.

[0038] Rather, antisense oligomers are used to alter the transcription process to increase the production of truncated, nonsense, or prematurely terminated proteins. Preferably, the present invention results in the skipping of exon 2 to induce a premature stop codon in exon 3. This can result in a mutant transcript that cannot be translated into a functional PTP1B protein.

[0039] Preferably, the antisense oligomer targets a splice site within the PTPN1 gene. The target site may also include some flanking sequences around the splice site.

[0040] The antisense oligomer may also or alternatively bind to the polyadenylation site. The target site may also be near the polyadenylation site, but may not overlap with the polyadenylation site, i.e., may instead cover the sequence upstream or downstream of the polyadenylation site; in these cases, the antisense oligomer may not specifically cover the polyadenylation site. The location near the polyadenylation site may be sufficient to prevent the ability of the cleavage agent to bind to the polyadenylation site.

[0041] According to a first aspect of the present invention, there is provided an antisense oligomer capable of binding to a selected target on a PTPN1 gene transcript and altering pre-mRNA splicing in the PTPN1 gene transcript or a portion thereof.

[0042] For example, in one embodiment of the present invention, antisense oligomers of 10 to 50 nucleotides are provided that contain targeting sequences complementary to regions near or within the splice and / or polyadenylation sites of the PTPN1 pre-mRNA.

[0043] The terms "antisense oligomer" and "antisense compound," as well as "antisense oligonucleotide" and "ASO," are used interchangeably and refer to a sequence of cyclic subunits, each having base-pairing portions linked by intersubunit linkages that allow the base-pairing portions to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing, forming a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunits are based on ribose or another pentose sugar, or, in a preferred embodiment, a morpholino group (see the description of morpholino oligomers below). The oligomer can have exact or near sequence complementarity to the target sequence. Sequence mutations near the ends of the oligomer are generally preferred over internal mutations. The terms "pre-RNA" and "pre-mRNA" are used interchangeably.

[0044] "Isolated" refers to a material that is substantially or essentially free from components that normally accompany it in nature. For example, as used herein, "isolated polynucleotide" or "isolated oligonucleotide" can refer to a polynucleotide that has been purified or removed from adjacent sequences in its naturally occurring state, such as a DNA fragment that has been removed from adjacent sequences in a genome. The term "isolate" with respect to cells refers to the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with a polynucleotide repeat disease). In the context of mRNA or protein, "isolate" refers to the recovery of mRNA or protein from a source, such as a cell.

[0045] An antisense oligomer can be said to be "directed" or "targeted" to the target sequence to which it hybridizes. In certain embodiments, the target sequence includes a region containing a splice site and / or polyadenylation site and surrounding regions. The target sequence is typically a region containing the AUG start codon of an mRNA, a translation suppressor oligomer, or a splice site or splice suppressor oligomer (SSO) of a preprocessed mRNA. A splice site target sequence can include an mRNA sequence having 1 to about 25 base pairs at its 5' end downstream of the normal splice acceptor junction of the preprocessed mRNA. Preferred target sequences are any region of a preprocessed mRNA that includes a splice site, is contained entirely within an exon-coding sequence, or spans the splice acceptor or donor site. More generally, an oligomer is said to "target" a biologically relevant target, such as a protein, virus, or bacterium, when it is targeted to the target nucleic acid in the manner described above.

[0046] As used herein, "sufficient length" refers to an antisense oligonucleotide that is complementary to at least 8, more typically 8 to 30, consecutive nucleobases in the target PTPN1 pre-mRNA. In some embodiments, a sufficiently long antisense oligonucleotide comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleobases in the target PTPN1 pre-mRNA. In other embodiments, a sufficiently long antisense oligonucleotide comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleobases in the target PTPN1 pre-mRNA. A sufficiently long antisense oligonucleotide has at least a minimum number of nucleotides capable of specifically hybridizing to exon 2. Preferably, oligonucleotides of sufficient length are about 10 to about 50 nucleotides in length, including oligonucleotides of 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, oligonucleotides of sufficient length are 10 to about 30 nucleotides in length. In another embodiment, oligonucleotides of sufficient length are 15 to about 25 nucleotides in length. In yet another embodiment, oligonucleotides of sufficient length are 20 to 30 or 20 to 50 nucleotides in length. In yet another embodiment, oligonucleotides of sufficient length are 22 to 28, 25 to 28, 24 to 29, or 25 to 30 nucleotides in length.

[0047] In certain embodiments, the antisense oligomer has sufficient sequence complementarity to the target RNA (e.g., pre-mRNA) to effectively block a region of the target RNA (i.e., the RNA where splicing factor binding site selection is regulated). In exemplary embodiments, such blockage of the PTPN1 pre-mRNA serves to regulate or alter splicing by masking binding sites for natural proteins that would otherwise regulate splicing and / or by altering the structure of the target RNA. In some embodiments, the target RNA is a target pre-mRNA (e.g., a PTPN1 gene pre-mRNA).

[0048] An antisense oligomer having sufficient sequence complementarity to a target RNA sequence to modulate splicing factor binding of the target RNA means that it has sufficient sequence to cause masking of the native protein binding site that would normally cause cleavage of the PTP1B protein and / or alter the three-dimensional structure of the target RNA.

[0049] The selected antisense oligomer can be shorter, e.g., about 12 bases, or longer, e.g., about 50 bases, and contain a small number of mismatches so long as the sequences are sufficiently complementary to modulate splicing factor binding upon hybridization to the target sequence, optionally forming an RNA antisense oligomer heteroduplex with a Tm of 45°C or greater.

[0050] Preferably, the antisense oligomer is selected from the group comprising the sequences set forth in Tables 1 and 4. Preferably, the antisense oligomer is selected from the table comprising SEQ ID NOs: 1 to 4, 10 to 15, 18 to 19, 23 to 25, 27, 29, 31 to 41, 42 to 57, or 69 to 75. Preferably, the antisense oligomer used in the present invention is selected from the table comprising SEQ ID NOs: 42 to 57, or 69 to 75. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 42, 46, 50, or 52. Preferably, the antisense oligomer results in exon skipping of exon 2.

[0051] In certain embodiments, the degree of complementarity between the target sequence and the antisense oligomer is sufficient to form a stable duplex. The region of complementarity 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 longer, such as 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. An antisense oligomer of approximately 16-17 bases is generally long enough to have a unique complementary sequence. In certain embodiments, a minimum length of complementary bases may be required to achieve the required binding Tm, as discussed herein.

[0052] In certain embodiments, oligonucleotides of 50 bases in length may be suitable, with at least a minimum number of bases, e.g., 10-12 bases, complementary to the target sequence. However, generally, enhanced or active uptake in cells is optimized with oligonucleotide lengths of less than about 30 bases. For phosphorodiamidate morpholino oligomer (PMO) antisense oligomers, as further described herein, the optimal balance between binding stability and uptake generally occurs with lengths of 18-25 bases. Included are antisense oligomers (e.g., PMO, PMO-X, PNA, LNA, 2'-OMe) 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.

[0053] In certain embodiments, antisense oligomers can be 100% complementary to target sequence, or can contain mismatches, for example, to accommodate mutations, as long as the heteroduplex formed between antisense oligomers and target sequence is stable enough to withstand the action of cellular nuclease and other degradation modes that can occur in vivo.Therefore, certain oligonucleotides can have about or at least about 70% sequence complementarity between oligonucleotides and 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.

[0054] Mismatches, if present, are typically less destabilizing toward the terminal regions of the hybrid duplex than toward the center. The number of mismatches tolerated depends on the length of the antisense oligomer, the proportion of G:C base pairs in the duplex, and the location of the mismatch(es) in the duplex, according to well-understood principles of duplex stability. While such antisense oligomers are not necessarily 100% complementary to the target sequence, they are effective in stably and specifically binding to the target sequence, thereby modulating splicing factor binding to the target pre-mRNA.

[0055] The stability of the duplex formed between an antisense oligomer and a target sequence is a function of the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of an oligonucleotide with respect to its complementary sequence RNA can be measured by conventional methods, such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154, pp. 94-107. In certain embodiments, the antisense oligomer can have a binding Tm with respect to its complementary sequence RNA above body temperature, preferably above about 45°C or 50°C. Tms in the range of 60-80°C or higher are also included.

[0056] Further examples of variants include antisense oligomers having about or at least about 70% sequence identity or homology, 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, over the entire length of any of SEQ ID NOs: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57, 69-73, or 75.

[0057] More specifically, antisense oligomers capable of binding to selected target sites and modulating or altering splicing in PTPN1 gene transcripts or portions thereof are provided. Antisense oligomers are preferably selected from those shown in Tables 1 and 4. Preferably, antisense oligomers are selected from the table containing SEQ ID NOs: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57, or 69. Preferably, antisense oligomers used in the present invention are selected from the table containing SEQ ID NOs: 42-57, or 69-75. More preferably, antisense oligomers used in the present invention are SEQ ID NOs: 42, 46, 50, or 52.

[0058] The antisense oligomer-induced splicing factor blockade of the present invention need not completely or substantially reduce the amount of PTP1B produced. [Table 1]

[0059] Preferably, the antisense oligomer is selected from the list comprising SEQ ID NOs: 1 to 4, 10 to 15, 18 to 19, 23 to 25, 27, 29, 31 to 41, 42 to 57, or 69 to 75. More preferably, the antisense oligomer used in the present invention is selected from the list comprising SEQ ID NOs: 42 to 57 or 69 to 75. Most preferably, the antisense oligomer used in the present invention is SEQ ID NO: 42, 46, 50, or 52.

[0060] How to use The present invention further provides a method for manipulating splicing factor binding in a PTPN1 gene transcript, comprising: Further provided is a method comprising the steps of: a) providing one or more of the antisense oligomers described herein and allowing the oligomer(s) to bind to a target nucleic acid site.

[0061] According to yet another aspect of the present invention, there is provided a splicing factor binding modification target nucleic acid sequence for PTPN1, comprising a DNA equivalent of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57, or 69-75, or a sequence complementary thereto. Preferably, the antisense oligomer used in the present invention is selected from the list comprising SEQ ID NOs: 42-57 or 69-75. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 42, 46, 50, or 52. Preferably, the antisense oligomer causes exon skipping of exon 2.

[0062] Designing antisense oligomers to completely mask splicing and / or polyadenylation sites may not be necessary to cause changes in the proportion of truncated, nonsense, or prematurely terminated proteins.Furthermore, the present inventors have discovered that the size or length of the antisense oligomer itself is not necessarily a major factor when designing antisense oligomers.For some targets, antisense oligomers as short as 20 bases can sometimes induce truncation modifications more efficiently than other longer (e.g., 25 base) oligomers directed to the same region.

[0063] More specifically, the antisense oligomer can be selected from those shown in Tables 1 and 4. The sequence is preferably selected from the group consisting of one or more of SEQ ID NOs: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57, or 69-75, and combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that possess or modulate RNA processing activity in PTPN1 gene transcripts. Preferably, the ASO used in the present invention is selected from the list including SEQ ID NOs: 42-57 or 69-75. More preferably, the antisense oligomer used in the present invention is SEQ ID NO: 42, 46, 50, or 52. Preferably, the antisense oligomer results in exon skipping of exon 2.

[0064] Antisense oligomers and DNA, cDNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other.Therefore, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or pairing, so that stable and specific binding occurs between oligomers and DNA, cDNA or RNA target.It is understood in the art that the sequence of antisense oligomers does not need to be 100% complementary to the sequence of its target sequence in order to be specifically hybridizable.Antisense oligomers are specifically hybridizable when the binding of compound to target DNA or RNA molecule interferes with the normal function of target DNA or RNA product, and there is a sufficient degree of complementarity to avoid the non-specific binding of antisense oligomers to non-target sequence under the conditions that specific binding is desired, i.e., under physiological conditions for in vivo assay or therapeutic treatment, and under the conditions that assay is performed for in vitro assay.

[0065] Selective hybridization can occur under low, medium, or high stringency conditions, but is preferably under high stringency. Those skilled in the art will recognize that hybridization stringency is influenced by conditions such as salt concentration, temperature, and organic solvents, in addition to base composition, length of complementary strands, and the number of nucleotide base mismatches between hybridizing nucleic acids. Stringent temperature conditions generally include temperatures above 30°C, typically above 37°C, preferably above 45°C, preferably at least 50°C, and typically 60°C to 80°C or higher. Stringent salt conditions are usually below 1000 mM, typically below 500 mM, and preferably below 200 mM. However, the combination of parameters is far more important than the measurement of any single parameter. An example of stringent hybridization conditions is 65°C and 0.1x SSC (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate pH 7.0). Thus, antisense oligomers of the present invention can include oligomers that selectively hybridize to the sequences provided in Table 1 and Table 4, SEQ ID NOs: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57 or 69-75.

[0066] At a given ionic strength and pH, Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Such hybridization can occur with "near" or "substantial" complementarity, as well as exact complementarity, of the antisense oligomer to the target sequence.

[0067] Typically, selective hybridization occurs when there is at least about 55% identity with the nucleotide of antisense oligomer over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, most preferably at least about 90%, 95%, 98% or 99% identity.As described, the length of homology comparison can be over a longer stretch, and in certain embodiments, it often over a stretch of at least about 9 nucleotides, usually at least about 12 nucleotides, more usually at least about 20, often 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, at least about 36 or more nucleotides.

[0068] 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 shown in the sequence listing 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 greater the homology required to achieve selective hybridization. As a result, when the antisense oligomer of the present invention consists of less than about 30 nucleotides, it is preferred that the percentage identity is greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96, 97, 98% or 99% compared to the antisense oligomer listed in the sequence listing herein. Nucleotide homology comparisons can be performed using sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research, 12, 387-395). In this way, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0069] The antisense oligomer of the present invention may have regions of reduced homology and regions of exact homology with the target sequence. The oligomer need not have exact homology over its entire length. For example, the oligomer may have a continuous stretch of at least 4 or 5 bases identical to the target sequence, preferably a continuous stretch of at least 6 or 7 bases identical to the target sequence, and more preferably a continuous stretch of at least 8 or 9 bases identical to the target sequence. The oligomer may have a stretch 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 stretches of the oligomer sequence may be intermittently identical to the target sequence. For example, the remaining sequence may have an identical base followed by a non-identical base followed by an identical base. Alternatively (or similarly), the oligomer sequence may have several stretches of identical sequence (e.g., 3, 4, 5, or 6 bases) interspersed with stretches of less than perfect homology. Such sequence mismatches preferably result in no or little loss of cleavage-modifying activity.

[0070] The term "modulate" or "modulates" optionally includes "increasing" or "decreasing" one or more quantifiable parameters by a defined and / or statistically significant amount. The terms "increase" or "increasing," "enhance" or "enhancing," or "stimulate" or "stimulating" generally refer to the ability of an antisense oligomer or composition to produce or cause a greater physiological response (i.e., a downstream effect) in a cell or subject compared to the response caused by neither the antisense oligomer nor a control compound.

[0071] "Enhance" or "enhancing," or "increase" or "increasing," or "stimulate" or "stimulating" generally refers to the ability of an antisense compound or composition to produce or cause a greater physiological response (i.e., a downstream effect) in a cell or subject compared to the response caused by either no antisense compound or a control compound. A measurable physiological response can include a decrease in expression of a functional form of the PTP1B protein, among other responses apparent from understanding in the art and the description herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) times the amount produced in the absence of the antisense compound (no drug) or by a control compound.

[0072] The term "decreasing" or "decrease" generally refers to the ability of an antisense oligomer or composition to produce or cause a decreased physiological response (i.e., a downstream effect) in a cell or subject, compared to the response caused by either the antisense oligomer or a control compound. The term "reducing" or "inhibiting" generally may refer to the ability of one or more antisense compounds of the invention to "reduce" a relevant physiological or cellular response, e.g., a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic field. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and may include a reduction in the symptoms or pathology of a PTP1B-associated condition. A "reduction" in response may be statistically significant compared to the response occurring without the antisense compound or control composition and may include a 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% reduction, including all integers in between.

[0073] The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a decrease in the amount of PTP1B protein. An "increased" or "enhanced" amount will generally be 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 (e.g., 500-fold, 1000-fold) (including all integers and decimal points above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the amount produced without the antisense oligomer (in the absence of agent) or by a control compound. The terms "reducing" or "inhibiting" may generally refer to the ability of one or more antisense oligomers or compositions to "reduce" the relevant physiological or cellular response, e.g., the symptoms of a disease or condition described herein, as measured according to routine techniques in the diagnostic arts. 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 the symptoms or pathology of diseases associated with T2DM, such as insulin resistance and leptin resistance, or diseases such as cancer. A "reduction" in response may be statistically significant compared to the response produced by no antisense oligomer or a control composition, and may include a 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% reduction (including all integers therebetween).

[0074] The length of the antisense oligomer can vary as long as it can selectively bind to the intended position in the pre-mRNA molecule. The length of such a sequence can be determined according to the selection procedure described herein. Generally, the antisense oligomer is about 10 to about 50 nucleotides in length. However, it can be understood that any length of nucleotide within this range can be used in this method. Preferably, the length of the antisense oligomer is 10 to 40, 10 to 35, 15 to 30, or 20 to 30 nucleotides, most preferably about 25 to 30 nucleotides. For example, the oligomer can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0075] As used herein, "antisense oligomer" or "ASO" refers to a linear sequence of nucleotides or nucleotide analogs whose nucleobases hybridize to a target sequence in RNA by Watson-Crick base pairing, forming an oligonucleotide:RNA heteroduplex within the target sequence. The terms "antisense oligomer," "antisense oligonucleotide," "oligomer," and "antisense compound" may be used interchangeably to refer to an oligonucleotide. The cyclic subunit may be based on a ribose or another pentose sugar, or, in certain embodiments, a morpholino group (see the description of morpholino oligonucleotides below). Peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and 2'-O-methyl (2'-OMe) oligonucleotides, among other antisense agents known in the art, are also contemplated.

[0076] In some embodiments, antisense oligonucleotides have the chemical composition of naturally occurring nucleic acid molecules, i.e., they do not contain modified or substituted bases, sugars or intersubunit linkages.

[0077] In preferred embodiments, the antisense oligonucleotides of the present invention are non-naturally occurring nucleic acid molecules or "oligonucleotide analogs." For example, non-naturally occurring nucleic acids can contain one or more non-natural bases, sugars, and / or intersubunit linkages, e.g., bases, sugars, and / or linkages that are modified or substituted relative to those found in naturally occurring nucleic acid molecules. Variations are described below. In some embodiments, non-naturally occurring nucleic acids contain more than one type of modification, e.g., sugar and base modifications, sugar and linkage modifications, base and linkage modifications, or base, sugar, and linkage modifications. For example, in some embodiments, the antisense oligonucleotides contain non-natural (e.g., modified or substituted) bases. In some embodiments, the antisense oligonucleotides contain non-natural (e.g., modified or substituted) sugars. In some embodiments, the antisense oligonucleotides contain non-natural (e.g., modified or substituted) intersubunit linkages. In some embodiments, the antisense oligonucleotides contain more than one type of modification or substitution, e.g., non-natural bases and / or non-natural sugars and / or non-natural intersubunit linkages.

[0078] Therefore, non-natural antisense oligomers include those with (i) modified backbone structures, for example, backbones other than the standard phosphodiester bond found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) modified sugar moieties, for example, morpholino moieties instead of ribose or deoxyribose moieties.Oligonucleotide analogs support bases that can hydrogen bond with standard polynucleotide bases through Watson-Crick base pairing, where the analog backbone presents the bases in a manner that allows such hydrogen bonding between the oligonucleotide analog molecule and the base in standard polynucleotide in a sequence-specific manner (for example, single-stranded RNA or single-stranded DNA).Preferred analogs have substantially uncharged phosphorus-containing backbones.

[0079] One method for generating antisense oligomers is methylation of the 2' hydroxyribose position, and incorporation of a phosphorothioate backbone produces molecules that superficially resemble RNA but are much more resistant to nuclease degradation, although those skilled in the art will recognize other forms of suitable backbones that may be used for purposes of the present invention.

[0080] To avoid degradation of the pre-RNA during duplex formation with the antisense oligomer, the antisense oligomer used in the present method can be adapted to minimize or prevent cleavage by endogenous RNase H. Antisense molecules that do not activate RNase H can be produced according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modifications that sterically hinder or prevent RNase H binding to duplex molecules containing the oligonucleotide as one of their members, and the structural modifications do not substantially hinder or prevent duplex formation. Because the portion of the oligonucleotide involved in duplex formation is substantially different from the portion involved in RNase H binding to it, numerous antisense molecules that do not activate RNase H are available. This property is highly desirable, as treatment of RNA with unmethylated oligomers in cells or crude extracts containing RNase H results in degradation of the pre-mRNA:antisense oligomer duplex. Any form of modified antisense oligomer that can bypass or cannot induce such degradation can be used in this method. Nuclease resistance can be achieved by modifying the antisense oligomer of the present 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.

[0081] An example of an antisense oligomer that is not cleaved by cellular RNase H when duplexed with RNA is a 2'-O-methyl derivative. Such 2'-O-methyl-oligoribonucleotides are stable in cellular environments and animal tissues, and their duplexes with RNA have higher Tm values ​​than their corresponding ribonucleotides or deoxyribonucleotides. Alternatively, at least one of the last 3'-terminal nucleotides of the nuclease-resistant antisense oligomer of the present invention may be fluorinated. Furthermore, the nuclease-resistant antisense oligomer of the present invention has phosphorothioate bonds between at least two of the last three terminal nucleotide bases, and preferably has phosphorothioate bonds between the last four 3'-terminal nucleotide bases.

[0082] Modified or regulated RNA splicing can also be achieved using alternative oligonucleotide chemical modifications (e.g., U.S. Patent No. 5,149,797). For example, antisense oligomers can be made using thiomorpholino oligomers (TMOs), phosphoramidate morpholino oligomers or phosphorodiamidate morpholino oligomers (PMOs), PMO-X, PPMOs, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and derivatives including alpha-L-LNA, 2'-amino LNA, 4'-methyl LNA, and 4'-O-methyl LNA, ethylene-bridged nucleic acids (ENAs) and their derivatives, phosphorothioate oligomers, tricyclo-DNA oligomers, etc. The oligomers may be selected from the list comprising 2'-O-methyl modified oligomers (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-fluoro, 2'-fluoroarabino (FANA), unlocked nucleic acid (UNA), hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), 2'-amino (2'-NH), 2'-O-ethyleneamine as a mixmer or gapmer or any combination of the above.

[0083] To further improve delivery efficacy, the above-mentioned modified nucleotides are often conjugated to the sugar or nucleobase moiety with fatty acids, lipids, cholesterol, amino acids, carbohydrates, polysaccharides, nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct antisense oligomers to alter splicing factor binding. Antisense oligomer-induced splicing factor binding modifications of PTPN1 gene transcripts generally use either oligoribonucleotides, PNAs, 2'OMe, or MOE-modified bases on phosphorothioate backbones. 2'OMe ASOs are used in oligo design, but despite their efficient in vitro uptake when delivered as cationic lipoplexes, these compounds are susceptible to nuclease degradation and are not considered ideal for in vivo or clinical use. When alternative chemical modifications are used to generate the antisense oligomers of the present invention, uracil (U) in the sequences provided herein may be replaced with thymine (T).

[0084] For example, such antisense molecules can be oligonucleotides in which at least one or all of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates, and fluorophoramidates. For example, every other internucleotide bridging phosphate residue can be modified as described. 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, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other nucleotide can be modified as described.

[0085] Specific examples of antisense oligonucleotides useful in the present invention include oligonucleotides containing modified backbones or non-natural intersubunit linkages.

[0086] Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleotides.

[0087] In other antisense molecules, both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0088] Modified oligonucleotides can also contain one or more substituted sugar moieties.Oligonucleotides can also contain nucleic acid base (often simply referred to in the art as "base") modification or substitution.The oligonucleotides containing modified or substituted bases include those in which one or more of the purine or pyrimidine bases that are most commonly found in nucleic acids are replaced with less common or unnatural bases.

[0089] Purine bases contain a pyrimidine ring fused to an imidazole ring. Adenine and guanine are the two most commonly found purine nucleobases in nucleic acids. These may be substituted with other naturally occurring purines, including, but not limited to, N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0090] Pyrimidine bases comprise a 6-membered pyrimidine ring.Cytosine, uracil and thymine are the most common pyrimidine bases found in nucleic acid.They can be substituted with 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 thymine base instead of uracil.

[0091] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and derivatives thereof, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super Examples of Super A, Super G, and Super T derivatives include N4-ethylcytosine, N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil, and degenerate or universal bases, such as 2,6-difluorotoluene, or bases that do not contain bases, such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, or pyrrolidine derivatives in which the ring oxygen is replaced with nitrogen (azaribose)). Examples of Super A, Super G, and Super T derivatives can be found in U.S. Patent No. 6,683,173 (Epoch Biosciences). cPent-G, cPent-AP, and Pr-AP have been shown to reduce immunostimulatory effects when incorporated into siRNA (Peacock H., et al. J. Am. Chem. Soc. 2011, 133, 9200). Pseudouracil is a naturally occurring isomerized version of uracil, which has a C-glycoside instead of the usual N-glycoside like uridine. Pseudouridine-containing synthetic mRNA may have an improved safety profile compared to uridine-containing mPvNA (see International Publication No. 2009127230).

[0092] 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. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C and are currently the preferred base substitution, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0093] In some embodiments, modified or substituted nucleobases are useful for facilitating the purification of antisense oligonucleotides.For example, in certain embodiments, antisense oligonucleotides can contain three or more (for example, 3, 4, 5, 6 or more) consecutive guanine bases.In certain antisense oligonucleotides, a string of three or more consecutive guanine bases can cause oligonucleotide aggregation, complicating purification.In such antisense oligonucleotides, one or more consecutive guanine bases can be replaced with inosine.The replacement of 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.

[0094] In one embodiment, another modification of antisense oligonucleotides comprises chemically linking one or more moieties or conjugates to the oligonucleotide to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.Such moieties include, but are not limited to, cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or lipid moieties such as adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0095] Not all positions of a given compound need to be uniformly modified, and in fact, two or more of the above-mentioned modifications can be incorporated into a single nucleoside in a single compound or oligonucleotide.The present invention also includes antisense oligonucleotides that are chimeric compounds.In the context of the present invention, " chimeric " antisense compounds or " chimeras " are antisense molecules, particularly oligonucleotides, that comprise two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., in the case of oligonucleotide compounds, nucleotides.These oligonucleotides typically comprise at least one region that the oligonucleotide is modified to provide additional regions for increasing resistance to nuclease degradation, increasing cellular uptake, and increasing binding affinity to target nucleic acid.

[0096] The antisense molecule used according to the present invention can be conveniently and routinely produced by the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California).One method for synthesizing oligonucleotide on modified solid support is described in U.S. Patent No. 4,458,066.

[0097] In another non-limiting example, such an antisense oligomer is a molecule 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, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other nucleotide can be modified as described.

[0098] While the antisense oligomers described above are preferred forms of antisense oligomers of the present invention, the present invention includes other oligomeric antisense molecules, including but not limited to oligomer mimetics as described below.

[0099] Another preferred chemical entity is phosphorodiamidate morpholino oligomer (PMO) oligomeric compounds that are not degraded by any known nucleases or proteases. These compounds are uncharged, do not activate RNase H activity when bound to RNA strands, and have been shown to exert persistent modulation of cleavage factor binding after in vivo administration (Summerton and Weller, Antisense Nucleic Acid Drug Development, 7, 187-197).

[0100] Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the 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. 5-methylcytosine substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications, have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C. In one embodiment, at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, the pyrimidine base being 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.

[0101] In one embodiment, the antisense oligonucleotides contain 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 oligonucleotides contain one or more 2,6-diaminopurine substitutions, alone or in combination with another modification.

[0102] In some embodiments, antisense oligonucleotides are chemically linked to one or more moieties, such as polyethylene glycol moieties, or conjugates, such as arginine-rich cell-penetrating peptides, which enhance the activity, cellular distribution, or cellular uptake of antisense oligonucleotides.In an exemplary embodiment, an arginine-rich polypeptide is covalently linked to the 3' or 5' end of the antisense compound at its N-terminal or C-terminal residue.Also in exemplary embodiments, the antisense compound is composed of morpholino subunits and a phosphorus-containing intersubunit bond that connects the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit.

[0103] In another aspect, the present invention provides an expression vector incorporating the above-described antisense oligonucleotide, for example, the antisense oligonucleotide of SEQ ID NOs: 1 to 81. In some embodiments, the expression vector is a modified retroviral or non-retroviral vector, such as an adeno-associated viral vector.

[0104] Another modification of the oligomers of the invention involves chemically attaching to the oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligomer. Such moieties include, but are not limited to, cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or lipid moieties such as adamantane acetic acid, palmityl moieties, myristyl, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0105] Cell-penetrating peptides have been added to phosphorodiamidate morpholino oligomers to enhance cellular uptake and nuclear localization. As shown in Jearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624–1629, various peptide tags have been shown to affect uptake efficiency and target tissue specificity. 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 shown herein, peptides have the ability to induce cell penetration within 100% of cells in a given cell culture population, enabling macromolecular transport within multiple tissues in vivo upon systemic administration.

[0106] Not all positions of a given compound need be uniformly modified; in fact, two or more of the above modifications may be incorporated into a single compound, or even into a single nucleoside within an oligomer.The present invention also includes antisense oligomers that are chimeric compounds.In the context of the present invention, a "chimeric" antisense oligomer or "chimera" refers to an antisense oligomer, particularly an oligomer, that comprises two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., in the case of an oligomeric compound, a nucleotide.These oligomers typically comprise at least one region that is modified to provide the oligomer or antisense oligomer with additional regions for increased resistance to nuclease degradation, increased cellular uptake, and increased binding affinity to target nucleic acid.

[0107] The activity of antisense oligomers and their variants can be assayed according to routine techniques in the art.For example, the form and expression level of the isoforms of the investigated RNA and protein can be evaluated by any of a variety of well-known methods for detecting isoforms and / or the expression of transcribed nucleic acid or protein.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 blot and / or nucleic acid array, fluorescent in situ hybridization for detecting intracellular RNA transcripts, nucleic acid amplification methods, immunological methods for detecting proteins, protein purification methods, and protein function or activity assays.

[0108] RNA expression levels can be assessed by preparing RNA / cDNA (i.e., transcribed polynucleotides) from cells, tissues, or organisms and hybridizing the RNA / cDNA with a reference polynucleotide that is the complement of the assayed nucleic acid or a fragment thereof. Optionally, the cDNA can be amplified using various polymerase chain reaction or in vitro transcription methods before hybridization with the complementary polynucleotide. Preferably, the cDNA is not amplified. The expression of one or more transcripts can also be detected using quantitative PCR to assess the expression level of transcript T1(s).

[0109] The present invention provides clinically relevant oligomer chemistries and delivery systems for directing antisense oligomers that modify splicing factor binding of PTPN1 gene transcripts, reducing full-length PTPN1 transcripts to therapeutic levels. 1) in vitro oligomer purification using cell lines with experimental evaluation of (i) modification of splicing factor binding target motifs, (ii) development of antisense oligomer length and oligomer cocktails, (iii) selection of chemical properties, and (iv) addition of cell-penetrating peptides (CPPs) to enhance oligomer delivery, and (v) encapsulation of antisense oligomers into liposomal nanoparticles to enhance oligomer delivery in vivo; and 2) detailed evaluation of novel approaches to reduce PTPN1 transcripts; This is achieved by:

[0110] Thus, it is demonstrated herein that PTPN1 RNA processing can be manipulated with specific antisense oligomers, thereby obtaining a functionally significant reduction in the amount of PTP1B protein, thereby alleviating the pathology of PTP1B-related diseases.

[0111] Preferably, the disease associated with PTP1B is (i) associated with the downregulation of insulin signaling in subjects, (ii) associated with the downregulation of leptin signaling pathway in subjects, and / or (iii) associated with the dysfunction of BDNF / TRKB pathway, such as in Rett syndrome, and / or (iv) associated with the growth, migration and invasion of cancer cells.For example, the disease can be T2DM and / or obesity.Furthermore, the disease can be Rett syndrome.Furthermore, the disease can be cancer.

[0112] The antisense oligomer used according to the present invention can be conveniently produced by the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California).One method for synthesizing oligomer on modified solid support is described in U.S. Patent No. 4,458,066.

[0113] Additionally or alternatively, any other means for such synthesis known in the art can be used.It is well known to use similar techniques to prepare oligomers such as phosphorothioates and alkylated derivatives.In one such automated embodiment, diethyl-phosphoramidite is used as starting material and can be synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0114] The antisense oligomers of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or genetic vector constructs designed to direct the in vivo synthesis of antisense oligomers. The molecules of the present invention may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or compound mixtures, such as liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations, to aid uptake, distribution, and / or absorption.

[0115] Also included are vector delivery systems capable of expressing the oligomeric PTPN1 targeting sequences of the invention, for example, 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, 42-57, or 69-75, as described herein. "Vector" or "nucleic acid construct" refers to a polynucleotide molecule, preferably a DNA molecule derived from a plasmid, bacteriophage, yeast, or virus, into which a polynucleotide can be inserted or cloned. The vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell, including a target cell or tissue or its progenitor cell or tissue, or may be capable of integrating into the genome of a defined host, such that the cloned sequence can be replicated. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any means for ensuring autonomous replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

[0116] Treatment method The antisense oligomers of the present invention can also be used as prophylactic or therapeutic agents, which can be utilized for the purpose of treating disease. Thus, in one embodiment, the present invention provides a therapeutically effective amount of an antisense oligomer that binds to a selected target in PTPN1 RNA and alters the splicing of the RNA described herein, in combination with a pharmaceutically acceptable carrier, diluent, or excipient.

[0117] An "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either in a single dose or as part of a series, effective to produce the desired therapeutic effect.

[0118] Accordingly, the present invention provides a pharmaceutical, prophylactic or therapeutic composition for treating, preventing or ameliorating the effects of a PTP1B-associated disease in a subject, the composition comprising: a) one or more antisense oligomers described herein; and b) one or more pharmaceutically acceptable carriers and / or diluents; and Includes.

[0119] Preferably, the disease associated with PTP1B is (i) a disease associated with the downregulation of insulin signaling in a subject, (ii) a disease associated with the downregulation of the leptin signaling pathway in a subject, and / or (iii) a disease associated with the dysfunction of the BDNF / TRKB pathway, such as in Rett syndrome, and / or (iv) a disease associated with the growth, migration and invasion of cancer cells. For example, the disease can be T2DM, obesity, Rett syndrome, or cancer.

[0120] Preferably, the antisense oligomers used in the present invention are selected from the list comprising: SEQ ID NOs: 1 to 4, 10 to 15, 18 to 19, 23 to 25, 27, 29, 31 to 41, 42 to 57, or 69 to 75, SEQ ID NOs: 42-57, or 69-75, SEQ ID NO: 42, 46, 50 or 52.

[0121] Preferably, the antisense oligomer results in exon skipping of exon 2.

[0122] The compositions may contain about 1 nM to 1000 nM of each desired antisense oligomer(s) of the present invention. Preferably, the compositions may contain 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(s) of the present invention.

[0123] The composition may comprise 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 of each of the desired antisense oligomer(s) of the invention.

[0124] The present invention further provides one or more antisense oligomers adapted to aid in the prophylactic or curative treatment of a disease or pathology associated with PTP1B, or the prevention or alleviation of symptoms thereof, in a form suitable for delivery to a subject.

[0125] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and, when administered to a subject, typically do not cause allergic or similar adverse reactions, such as stomach upset. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990).

[0126] Pharmaceutical Composition One aspect of the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more antisense oligomers of the present invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may include diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, as well as additives such as surfactants, solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Materials can be incorporated into liposomes or microparticle preparations of polymeric compounds such as polylactic acid and polyglycolic acid. Hyaluronic acid may also be used. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the present invention. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pp. 1435-1712, incorporated herein by reference. The compositions may be prepared in liquid form, or may be a dry powder, such as lyophilized form.

[0127] It will be understood that the pharmaceutical composition provided in accordance with the present invention can be administered by any means known in the art.Preferably, the pharmaceutical composition for administration is administered by injection, orally, topically, or via pulmonary or nasal routes.Antisense oligomers are more preferably delivered by intravenous, intraarterial, intraperitoneal, intramuscular or subcutaneous administration routes.Appropriate routes can be determined by those skilled in the art depending on the condition of the subject being treated.Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are some non-limiting sites where antisense oligomers can be introduced.Direct CNS delivery can be used, for example, intracerebral venules or intrathecal administration can be used as an administration route.

[0128] Formulations for topical administration include those in which the oligomer of the present disclosure is mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negatively charged (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other administration, the oligomer of the present disclosure may be encapsulated in a liposome or may be complexed with a cationic liposome. Alternatively, the oligomer may be complexed with a lipid, particularly a cationic lipid. Fatty acids and esters, their pharmaceutically acceptable salts, and their uses are further described in US Pat. No. 6,287,860 and / or US patent application Ser. No. 09 / 315,298, filed May 20, 1999.

[0129] In certain embodiments, the antisense oligomers of the present disclosure can be delivered by transdermal methods (e.g., by incorporating the antisense oligomer in an emulsion, for example, with such antisense oligomers optionally packaged in liposomes). Such transdermal and emulsion / liposome-mediated delivery methods are described in the art for the delivery of antisense oligomers, for example, in U.S. Patent No. 6,965,025.

[0130] The antisense oligomers described herein can also be delivered via implantable devices, the design of which is an art-recognized process, for example, by the synthetic implant designs described in U.S. Patent No. 6,969,400.

[0131] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, and binders may be desirable. Oral formulations are those in which the oligomers of the present 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, the present disclosure provides combinations of penetration enhancers, such as fatty acids / salts in combination with bile acids / salts. An exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The oligomers of the present disclosure may be orally delivered in granular form, including spray-dried particles, or may be complexed to form microparticles or nanoparticles. Oligomer complexing agents and their uses are further described in U.S. Patent No. 6,287,860. Oral formulations of oligomers and their preparation are described in detail in U.S. Patent Nos. 6,887,906, 09 / 315,298, and / or U.S. Patent Application Publication No. 20030027780, filed May 20, 1999.

[0132] Compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions which 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.

[0133] The delivery of therapeutically useful amounts of antisense oligomers can be achieved by previously published methods. For example, intracellular delivery of antisense oligomers can be achieved by a composition comprising a mixture of antisense oligomers and an effective amount of block copolymers. An example of this method is described in US Patent Application US20040248833. Other methods for delivering antisense oligomers to the nucleus are described in Mann CJ et al. (2001) Proc. Natl. Acad. Science, 98(1)42-47 and Gebski et al. (2003) Human Molecular Genetics, 12(15):1801-1811. Methods for introducing nucleic acid molecules into cells as naked DNA or via expression vectors complexed with lipid carriers are described in US Patent No. 6,806,084.

[0134] In certain embodiments, the antisense oligomers of the present invention and therapeutic compositions comprising them can be delivered by transdermal methods (e.g., by incorporating the antisense oligomer in an emulsion, for example, with such antisense oligomers optionally packaged in liposomes). Such transdermal and emulsion / liposome-mediated delivery methods are described in the art for the delivery of antisense oligomers, for example, in U.S. Patent No. 6,965,025.

[0135] It may be desirable to deliver antisense oligomers in colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, liposomes, or liposomal formulations. These colloidal dispersion systems can be used to prepare therapeutic pharmaceutical compositions.

[0136] Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo.These formulations may have net cationic, anionic, or neutral charge characteristics, and may have properties that are useful for in vitro, in vivo, and ex vivo delivery methods.Large unilamellar vesicles have been shown to be able to encapsulate a significant proportion of aqueous buffer containing large macromolecules.RNA and DNA can be encapsulated in the aqueous interior and delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci. 6:77, 1981).

[0137] For liposomes to be effective gene transfer vehicles, they must possess the following characteristics: (1) highly efficient encapsulation of the desired antisense oligomer without impairing its biological activity, (2) preferential and substantial binding to target cells compared with non-target cells, (3) highly efficient delivery of the aqueous contents of the vesicles to the cytoplasm of target cells, and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988). Liposomes are typically composed of phospholipids, particularly high-phase-transition-temperature phospholipids, combined with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are believed to entrap DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA into cells.

[0138] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in an extended circulation life compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0139] The antisense oligomers described herein can also be delivered via implantable devices. The design of such devices is an art-recognized process, involving, for example, the synthetic implant designs described in U.S. Patent No. 6,969,400, the contents of which are incorporated herein by reference in their entirety.

[0140] Antisense oligomers can be introduced into cells using techniques recognized in the art (for example, transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and non-viral vectors, and other means known in the art).The delivery method selected depends at least on the cells to be treated and the location of the cells, and will be clear to those skilled in the art.For example, localization can be achieved by liposomes with specific markers on the surface to guide liposomes, direct injection into the tissue containing target cells, specific receptor-mediated uptake, etc.

[0141] 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, permeabilization (e.g., streptolysin-O permeabilization, anionic peptide permeabilization), electroporation, and various non-invasive, non-endocytic delivery methods known in the art (see Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, incorporated by reference in its entirety).

[0142] Antisense oligomer can also be combined with other pharmaceutically acceptable carriers or diluents to produce pharmaceutical compositions.Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline.Composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.

[0143] The routes of administration described are intended as a guide only, as one skilled in the art can readily determine the optimum route of administration and any dosage for any particular animal and condition.

[0144] Several approaches have been attempted to introduce functional new genetic material into cells, both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl. 5074S-5079S), into non-retroviral vectors (Rosenfeld et al. (1992) Cell, 68:143-155; Rosenfeld et al. (1991) Science, 252:431-434), or delivery of 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, 252:431-434). Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), coupling to ligand-specific cation transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or using naked DNA expression vectors (Nabel et al. (1990) supra; Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues results in only localized expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra).Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) reported lung-only in vivo transfection of mice after intravenous or intratracheal administration of DNA-liposome complexes. Examples of reviews of 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.

[0145] The antisense oligomers of the present invention encompass any pharmaceutically acceptable salts, esters or salts of such esters, or any other compounds that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to an animal, including a human. Thus, by way of example, the present disclosure also relates to prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.

[0146] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present invention, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. In the case of oligomers, preferred examples of pharmaceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, and polyamines such as spermine and spermidine; (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid; (c) salts with organic acids such as 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, and polygalacturonic acid; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention can be administered in various ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (including ocular and mucosal, and rectal administration), pulmonary (e.g., by inhalation or insufflation of powder or aerosol) (including nebulizer, intratracheal, intranasal, epidermal, and transdermal), oral, or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, for example, intrathecal or intraventricular, administration.Oligomers with at least one 2'-O-methoxyethyl modification are considered particularly useful for oral administration.Preferably, antisense oligomers are delivered via subcutaneous or intravenous routes.

[0147] The pharmaceutical preparation of the present invention can be conveniently provided in unit dosage form and can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining the active ingredient with pharmaceutical carrier(s) or excipient T1(s). Generally, the preparation is prepared by uniformly and intimately mixing the active ingredient with liquid carrier(s) or finely divided solid carrier(s), or both, and then, if necessary, shaping the product.

[0148] Administration In one embodiment, the antisense oligomer is administered in an amount and manner effective to produce a peak blood concentration of the antisense oligomer of at least 200-400 nM. Typically, one or more doses of the antisense oligomer are administered, generally at regular intervals, over a period of about 1-2 weeks. A preferred dose for oral administration is about 1 mg-1000 mg of oligomer per 70 kg of body weight. In some cases, a dose of more than 1000 mg of oligomer per subject may be required. For intravenous administration, a preferred dose is about 0.5 mg-1000 mg of oligomer per 70 kg of body weight. For intravenous or subcutaneous administration, the antisense oligomer may be administered at a dose of about 120 mg / kg daily or weekly.

[0149] Antisense oligomer can be administered at regular intervals for a short period, for example, every day for 2 weeks or less.However, in some cases, oligomer can be administered intermittently for a longer period.Can be administered subsequently or simultaneously with the administration of antibiotics or other curative treatment.Treatment regimen can be adjusted (dosage, frequency, route etc.) as indicated based on the results of immunoassay, other biochemical tests and physiological tests of the subject under treatment.

[0150] Administration depends on the severity and responsiveness of the disease state being treated, with the course of treatment lasting from several days to several months, or until a cure is achieved or a diminution of the disease state is achieved. Optimal administration schedules can be calculated from measurements of drug accumulation in the subject's body. Those skilled in the art can easily determine optimal dosages, administration methods, and repetition rates. Optimal dosages may vary depending on the relative potency of individual oligomers and can usually be estimated based on the EC50 values ​​found to be effective in in vitro and in vivo animal models. Generally, dosages range from 0.01 μg to 100 g per kg of body weight and may be administered once or more daily, weekly, monthly, or yearly, or once every 2 to 20 years. Those skilled in the art can easily estimate repetition rates for administration based on measured residence times and drug concentrations in body fluids or tissues. After successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent recurrence of the disease state, in which case the oligomer is administered at a maintenance dose ranging from 0.01 μg to 100 g per kg of body weight, one or more times daily up to once every 20 years.

[0151] Effective in vivo therapeutic regimens using the antisense oligomers of the present invention may vary according to the duration, dose, frequency, and route of administration, as well as the condition of the subject being treated (i.e., prophylactic administration versus administration in response to a local or systemic infection). Thus, such in vivo treatments often require monitoring with tests appropriate for the particular type of disorder being treated, and corresponding adjustments of the dose or therapeutic regimen to achieve optimal therapeutic results.

[0152] Treatment can be monitored, for example, by common indicators of disease known in the art. As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or cell refers to any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and can be performed prophylactically or after the onset of a pathological event or contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with PTP1B, and can include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment, which can be aimed at reducing the rate of progression of the 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 complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0153] As used herein, a "subject" includes any animal that can be treated with the antisense compounds of the present invention, or that exhibits or is at risk of exhibiting any symptoms associated with these conditions (e.g., downregulation of insulin signaling, downregulation of the leptin signaling pathway, reduced cancer cell growth, migration, and invasion). Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, guinea pigs), farm animals, livestock, or pets (e.g., cats and dogs). Non-human primates and, preferably, human subjects are included.

[0154] The effectiveness of the in vivo administration antisense oligomer of the present invention can be determined from biological samples (tissue, blood, urine, etc.) collected from subject before, during and after administration of antisense oligomer.The assay of such sample can include: (1) using procedures known to those skilled in the art, for example, electrophoresis gel mobility assay, to monitor the presence or absence of heteroduplex formation between target sequence and non-target sequence; (2) monitoring the amount of mutant RNA compared with reference normal RNA or protein, which is determined by standard techniques such as RT-PCR, Northern blotting, ELISA or Western blotting.

[0155] Nuclear oligomer delivery is a major challenge for antisense oligomers. Different cell-penetrating peptides (CPPs) localize PMOs, or in certain embodiments, TMOs, to varying degrees under different conditions and cell lines, and novel CPPs have been evaluated by the inventors for their ability to deliver PMOs to target cells. The terms CPP or "cellular uptake-enhancing peptide moiety" are used interchangeably and refer to cationic cell-penetrating peptides, also known as "transport peptides," "carrier peptides," or "peptide transduction domains." As demonstrated herein, the 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, enabling macromolecular transport within multiple tissues in vivo upon systemic administration. CPPs are well known in the art and are disclosed, for example, in U.S. Patent Application No. 2010 / 0016215, the entire contents of which are incorporated by reference.

[0156] Thus, the present invention provides an antisense oligomer of the present invention in combination with a cell-penetrating peptide for the preparation of a therapeutic pharmaceutical composition.

[0157] According to yet a further aspect of the present invention, there is provided one or more antisense oligomers as described herein for use in antisense oligomer-based therapy. Preferably, the therapy is for a disease associated with PTP1B.

[0158] Preferably, the disease associated with PTP1B is (i) associated with the downregulation of insulin signaling in subjects, (ii) associated with the downregulation of leptin signaling pathway in subjects, and / or (iii) associated with the dysfunction of BDNF / TRKB pathway, such as in Rett syndrome, and / or (iv) associated with the growth, migration and invasion of cancer cells.For example, the disease can be T2DM and / or obesity.Alternatively, the disease can be Rett syndrome.Alternatively, the disease can be cancer.

[0159] More specifically, the antisense oligomer may be 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, 42-57, or 69-75, and combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that possess or modulate pre-RNA processing activity in PTPN1 gene transcripts. More preferably, the antisense oligomer used in the present invention is selected from the list comprising SEQ ID NOS: 42-57 or 69-75. Most preferably, the antisense oligomer used in the present invention is SEQ ID NOS: 42, 46, 50, or 52. Preferably, the antisense oligomer results in exon skipping of exon 2.

[0160] The present invention also extends to combinations of two or more antisense oligomers capable of binding to a selected target and altering the splicing of the PTPN1 gene transcript. The combination may be a cocktail of two or more antisense oligomers, and the construct comprises two or more antisense oligomers linked together for use in antisense oligomer-based therapy.

[0161] The present invention provides a method for treating, preventing, or ameliorating the effects of a disease associated with PTP1B, comprising: a) administering to a subject an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers described herein.

[0162] The present invention further provides a method for treating, preventing, or alleviating a disease associated with PTP1B, comprising: a) administering to a subject an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers described herein; Provided is a method, wherein the disease related to PTP1B is (i) related to the downregulation of insulin signaling in a subject, (ii) related to the downregulation of leptin signaling pathway in a subject, (iii) related to the dysfunction of BDNF / TRKB pathway in Rett syndrome, etc., and / or (iv) related to the growth, migration and invasion of cancer cells.For example, the disease can be T2DM and / or obesity.Alternatively, the disease can be Rett syndrome.Alternatively, the disease can be cancer.

[0163] Preferably, treatment is used to develop non-functional, truncated, or nonsense PTP1B proteins. Reduction of PTP1B levels is preferably achieved by reducing the amount of full-length transcript levels by binding to splice sites, resulting in exon skipping and / or altering pre-mRNA splicing factor binding in the PTPN1 gene transcript or portions thereof.

[0164] A reduction in PTP1B may preferably result in a reduction in the amount, duration or severity of symptoms of diseases associated with (i) downregulation of insulin signaling, (ii) downregulation of the leptin signaling pathway, such as in T2DM and / or obesity, and / or (iii) dysfunction of the BDNF / TRKB pathway, such as in Rett syndrome, and / or (iv) reduced cancer cell growth, migration and invasion.

[0165] According to another aspect of the present invention, there is provided the use of one or more antisense oligomers described herein in the manufacture of a medicament for modulating or controlling a disease associated with PTP1B.

[0166] The present invention also provides the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for the treatment of a disease associated with PTP1B.

[0167] Provided is the use of a purified and isolated antisense oligomer as described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with PTP1B.

[0168] Preferably, the antisense oligomers used in the present invention are selected from the list comprising: SEQ ID NOs: 1 to 4, 10 to 15, 18 to 19, 23 to 25, 27, 29, 31 to 41, 42 to 57, or 69 to 75, SEQ ID NOs: 42-57, or 69-75, SEQ ID NO: 42, 46, 50 or 52.

[0169] Preferably, the antisense oligomer results in exon skipping of exon 2.

[0170] According to a still further aspect, the present invention extends to cDNA or cloned copies of the antisense oligomer sequences of the invention, and to vectors containing the antisense oligomer sequences of the invention. The present invention further extends to cells containing such sequences and / or vectors.

[0171] The present invention also provides a kit for treating, preventing, or alleviating a PTP1B-related disease in a subject, the kit comprising at least an isolated or purified antisense oligomer for modulating pre-mRNA splicing factor binding in a PTPN1 gene transcript or portion thereof, packaged in a suitable container, together with instructions for use thereof.

[0172] In a preferred embodiment, the kit may contain at least one antisense oligomer (SEQ ID NOS: 1-4, 10-15, 18-19, 23-25, 27, 29, 31-41, 42-57, or 69-75) as described herein or as shown in Tables 1 and 4, or a cocktail of antisense oligomers as described herein. The kit may also include peripheral reagents such as buffers, stabilizers, etc.

[0173] Thus, there is provided a kit for treating, preventing or alleviating a disease associated with PTP1B 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, 42-57 or 69-75 as set forth in Tables 1 and 4, and combinations or cocktails thereof, packaged in a suitable container, together with instructions for use thereof.

[0174] Also provided is a kit for treating, preventing, or ameliorating a PTP1B-associated disease in a subject, the kit comprising at least an antisense oligomer 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, 42-57, or 69-75, and combinations or cocktails thereof, packaged in a suitable container, together with instructions for use thereof.

[0175] Preferably, the disease associated with PTP1B is (i) associated with the downregulation of insulin signaling in subjects, (ii) associated with the downregulation of leptin signaling pathway in subjects, and / or (iii) associated with the dysfunction of BDNF / TRKB pathway such as in Rett syndrome, and / or (iv) associated with the growth, migration and invasion of cancer cells.For example, the disease can be T2DM and / or obesity.Alternatively, the disease can be Rett syndrome.Alternatively, the disease can be cancer.

[0176] The contents of the kit can be lyophilized, and the kit can further include a solvent suitable for reconstitution of the lyophilized components. Individual components of the kit can be packaged in separate containers, and associated with such containers can be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by the agency of the manufacture, use, or sale for human administration.

[0177] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example, a sterile aqueous solution. For in vivo use, the expression construct can be formulated into a pharmaceutically acceptable injectable composition. In this case, the container means can itself be an inhalant, syringe, pipette, eye dropper, or other such device, from which the formulation can be applied to the affected area of ​​an animal, such as the lungs, injected into an animal, or applied to and mixed with other components of the kit.

[0178] The components of the kit may also be provided in a dried or lyophilized form. When reagents or components are provided in a dried form, reconstitution is usually achieved by adding a suitable solvent. It is also contemplated that the solvent may be provided in a separate container. Regardless of the number or type of container, the kit of the present invention may also include or be packaged with a device to assist in the injection / administration or placement of the final combined composition into the animal's body. Such a device may be an inhalant, syringe, pipette, forceps, a measured spoon, an eyedropper, or any such medically approved delivery vehicle.

[0179] Those skilled in the art will appreciate that the above methods have broad application for identifying antisense oligomers suitable for use in the treatment of many other diseases.

[0180] The antisense oligomers of the invention may also be used in combination with alternative therapies, such as drug therapy.

[0181] Thus, the present invention provides a method for treating, preventing, or alleviating the effects of a PTP1B-associated disease, in which an antisense oligomer of the present invention is administered sequentially or simultaneously with another alternative therapy associated with the treatment, prevention, or alleviation of the effects of a PTP1B-associated disease.

[0182] If the disease is related to insulin resistance, T2DM, leptin resistance or obesity, alternative therapies include insulin and insulin mimetics, drugs that increase insulin release (amylin mimetics such as pramlintide), sodium glucose transporter 2 inhibitors, e.g. canagliflozin, incretin mimetics [GLP-1 agonists] such as exenatide or liraglutide, dipeptidyl peptidase 4 inhibitors, e.g. saxagliptin, sitagliptin or linagliptin, sulfonylureas, e.g. glyburide, glipizide, glimepiride, chlorpropamide, tolazamide, gliquidone , glibenclamide, gliclazide, acetohexamide or tolbutamide, glinides such as nateglinide or repaglinide), drugs that decrease the absorption of sugar from the intestine (e.g., acarbose, voglibose and miglitol), drugs that prevent the reabsorption of filtered glucose by the kidneys (e.g., dapagliflozin and canagliflozin), drugs that make the body more sensitive to insulin (e.g., metformin, ciglitazone, troglitazone, rosiglitazone and pioglitazone), dietary changes combined with regular exercise, surgery to increase weight loss.

[0183] If the condition is related to Rett syndrome, alternative therapies can be chosen from a list including physical therapy / hydrotherapy to improve and maintain mobility and balance, occupational therapy to improve or maintain hand use, speech and language therapy to improve social interactions, feeding assistance to strengthen bones and slow scoliosis, physical assistance to correct scoliosis and coordinate hand movements, medication to reduce breathing problems, eliminate abnormal heart rhythm problems, reduce indigestion and constipation, and control seizures.

[0184] If the disease is cancer-related, alternative therapies may be selected from a list including chemotherapy, radiation therapy, surgery to remove solid tumors, and immunotherapy.

[0185] overview Throughout this specification, unless the context clearly indicates otherwise, the word "comprise" or variations of "comprises" or "comprising" will be understood to mean the inclusion of the specified element or group of elements, but not the exclusion of other elements or groups of elements.

[0186] Throughout this specification, unless the context clearly indicates otherwise, the word "comprise" or variations thereof such as "comprises" or "comprising" will be understood to mean the inclusion of the specified element or group of elements, but not the exclusion of other elements or groups of elements.

[0187] Other definitions of selected terms used herein are set forth in the Detailed Description of the Invention and apply throughout. Unless otherwise defined, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0188] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention encompasses all such variations and modifications. The invention includes all steps, features, combinations, and compounds referred to or shown in this specification, individually or collectively, and any combination of steps or features, or any two or more thereof.

[0189] Each document, reference, patent application, or patent cited herein is expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this document. It is solely for the sake of brevity that documents, references, patent applications, or patents cited herein are not repeated here.

[0190] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described herein or described in any document incorporated by reference herein are incorporated by reference and may be used in the practice of this invention.

[0191] The invention described herein may include one or more ranges of values ​​(e.g., concentrations). A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and values ​​adjacent to the range that produce the same or substantially the same result as the values ​​immediately adjacent to the values ​​defining the boundaries for the range.

[0192] The following examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. These examples are included solely for the purpose of illustrating the present invention. They are not to be construed as limitations on the broad summary, disclosure, or description of the invention above. Without further clarification, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. In the above and following examples, all temperatures are set forth uncorrected in degrees Celsius, and all parts and percentages are by weight unless otherwise noted. [Example]

[0193] Further features of the present invention will be more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the above broad summary, disclosure, or description of the invention.

[0194] Example 1: Design and synthesis of antisense oligonucleotides Forty-one ASOs (AOs) targeting human PTPN1 or mouse Ptpn1 transcripts were designed and synthesized as 2'-OMe nucleoside 3'-thiophosphates, as shown in Table 1. All of these AOs were transfected into Huh-7 and / or HepG2 cell lines (human hepatocellular carcinoma cell lines), IHH cell lines, normal human liver cell lines, and / or AML-12 cell lines, and normal mouse liver cell lines. The results showed that AOs 1-4, 10-15, 18, 19, 23-25, 27, 29, 31-41 could induce exon skipping (Figures 2-5, 8, 10-15, 17-19).

[0195] Human PTPN1 exon-2-targeting AOs (AO1, AO31-36) showed excellent exon-2 skipping effects when transfected into liver-related cell lines such as Huh-7 (Figure 2, Figure 5, Figure 13B), HepG2 (Figure 8, Figure 10, Figure 11, Figure 13A, Figure 14A), IHH (Figure 12, Figure 13C, Figure 14B, Figure 15), and even mouse AML-12 (Figure 18, Figure 19). To further evaluate the exon-2 skipping effect of AOs in mice, mouse Ptpn1 exon-2-targeting AOs (AO37-41) were transfected into both HepG2 cells (Figure 17) and AML-12 cells (Figure 18, Figure 19) to induce efficient exon-2 skipping. AOs (AO1, AO32-36) were found to be highly efficient in inducing human PTPN1 exon-2 skipping. Furthermore, AO33, PTPN1 1E2A(+5+29) (Diabexa-2), showed the best exon-2 skipping efficiency (Figures 12-14). In addition, its murine form or version, AO41, Ptpn1 1E2A(+5+29), also induced the highest percentage of exon-2 skipping in the murine AML-12 cell line (Figures 18 and 19), compared with other murine Ptpn1 exon-2 targeting AOs (AO37-40), even in the human HepG2 cell line (Figure 17).

[0196] General synthetic procedure Phosphorothioated (PS) 2'-O-methyl (2'OMe) AOs (Table 1) were designed and synthesized in-house using standard phosphoramidite chemistry on a 1 μmol scale using an ABI Expedite® 8909 Nucleic Acid Synthesis System (Applied Biosystems) or a GE AKTA Oligopilot 10 synthesizer (GE Healthcare Life Science). The synthesized AOs were deprotected and cleaved from the solid support by treatment with ammonium hydroxide (NH4OH) at 55°C for 8 hours. The crude AOs were then desalted using an Illustra NAP-10 column (GE Healthcare). The purified oligonucleotides were then verified by polyacrylamide gel electrophoresis.

[0197] Cell culture and transfection of ASOs into cells The human hepatoma cell line, Huh-7, was obtained from the American Type Culture Collection (ATCC). Another human hepatoma cell line, HepG2, was obtained from the European Collection of Authenticated Cell Cultures (ECACC). The Huh-7 cell line was cultured in 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific), and the HepG2 cell line was cultured in 10% FBS Eagle's minimum essential medium (ATCC). Both the 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 hepatoma cell lines were cultured at 55°C in a humidified atmosphere of 5% CO2. Cells were cultured to reach 70-90% confluency and then transfected at 5.0 × 10 cells per well for 24 hours before transfection. 4Cells were seeded into 24-well plates (Thermo Fisher Scientific) at a density of 1000 (cells / mL). The next day, for screening purposes, AO was transfected at a concentration of 400 nM using RNAiMAX reagent according to the original or modified manufacturer's protocol (the different modified transfection protocols are based on the manufacturer's instructions and are shown in Figure 6). 24 hours after transfection, cells were harvested for RNA extraction.

[0198] RNA extraction and RT-PCR RNA was extracted from transfected cells using Direct-zol™ RNA MinPrep Plus with TRI Reagent (Zymo Research) according to the manufacturer's instructions. Human PTPN1 exon-2 skipping products (product size: 639 bp) and non-skipped products (product size: 730 bp), and mouse Ptpn1 exon-2 skipping products (product size: 493 bp) and non-skipped products (product size: 584 bp) were amplified using a SuperScript® III One-Step RT-PCR Kit (Thermo Fisher Scientific) with the human PTPN1 primer pair (PTP1B_Ex1F: 5′-GTG ATG CGT AGT TCC GGC TG-3′, PTP1B_Ex6R: 5′-CAG GGA CTC CAA AGT CAG GC-3′) or the mouse Ptpn1 primer pair (Ptpn1_B_Ex1F: 5′-AGA TCG ACA AGG CTG GGA AC-3′, Ptpn1_B_Ex6R: 5′-TGA GCC TGA CTC TCG GAC TT-3′). Briefly, 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. PCR products were then separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized using a Fusion Fx gel documentation system. Densitometry was performed using ImageJ software.

[0199] Sequencing The bandstab technique was performed according to the guidelines of Anthony and James (1992). The bandstab samples were then amplified with the same primer set as above using the AmpliTaq Gold® DNA Polymerase Kit (Thermo Fisher Scientific). Briefly, 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. PCR products were verified on a 2% agarose gel and sent to the Australian Genome Research Facility (AGRF) for Sanger sequencing using both the forward and reverse primers described above.

[0200] Example 2: Evaluation of exon-2 skipping and full-length transcript knockdown efficacy The sequences of PTPN1 1E2A(+1+25) (AO1) and PTPN1 1E2A(+3+27) (AO32) are similar to one of the AOs previously described by Ionis Pharmaceuticals: PTPN1 1E2A(+1+20) (ISIS 107773). AO1 and AO32 have 2'-OMePS structures, whereas ISIS 107773 has a 5-10-5 MOE (2'-O-methoxyethyl) gapmer structure. A comparison was made between the 2'OMePS forms of AO1, AO32, AO36, AO32, and ISIS 107773, and the 5-10-5 MOE gapmer form of ISIS 107773, in terms of their ability to induce PTPN1 exon-2 skipping (Figures 8, 10, 13, and Table 2). The data showed that AO33 (Diabexa-2) best performed AO in inducing PTPN1 exon-2 skipping and / or full-length transcript knockdown. Furthermore, the sequence similarity of AO33-36 to ISIS 107773 is less than 70% (Table 3). [Table 2] [Table 3]

[0201] The ability of AOs (AO1, AO31-36) to induce PTPN1 exon-2 skipping was confirmed. For example, the ability of AO1 to induce exon-2 skipping was confirmed by Sanger sequencing (Figure 9).

[0202] Example 3. Evaluation of dose-dependent exon-2 skipping and full-length transcript knockdown efficacy Different concentrations (400, 200, 100, 50, 25, 12.5 nmol) of human PTPN1 or mouse Ptpn1 exon-2 targeting AO were transfected into different types of liver-related cells, and dose-dependent effects were observed. For example, AO1 induced efficient exon-2 skipping in HepG2 cells in a dose-dependent manner (Fig. 11), AO33 (Diabexa-2) induced efficient exon-2 skipping in both HepG2 cells and IHH cells in a dose-dependent manner (Fig. 14), and AO38 (the mouse version of AO32) and AO41 (the mouse version of AO33) induced efficient exon-2 skipping in mouse AML-12 cells in a dose-dependent manner (Fig. 19C, Fig. 19D). All these results above confirmed that PTPN1 exon-2 targeting AOs (AO1, AO32-36), most preferably AO33 (Diabexa-2), PTPN1 1E2A (+5+29), induced significant exon-2 skipping of PTPN1 transcripts, and thus these AOs could potentially induce a decrease in functional PTP1B protein production.

[0203] The results clearly demonstrated that AO33 (Diabexa-s), PTPN1 1E2A(+5+29), had better human PTPN1 exon-2 skipping and non-skipped product knockdown efficiencies than other exon-2-targeting AOs, including AO1, 32, 34-36, and both the 2'-OMePS and 5-10-5 MOE gapmer forms of ISIS 107773 (Figures 12-14). Furthermore, the mouse form of AO33 (Diabexa-2), i.e., AO41, showed the best mouse Ptpn1 exon-2 skipping efficacy compared to other Ptpn1 exon-2 targeting AOs (AO37-40) (Figures 17-19).

[0204] Example 4. Synthesis of modified ASO We synthesized and evaluated phosphorodiamidate morpholino oligomer (PMO) forms of AO targeting exon 2. For example, the PMO form of AO33 (Diabexa-2) was transfected into IHH cells by nucleofection and showed efficient PTPN1 exon 2 skipping in a dose-dependent manner (Figure 15). All of the above results confirmed that AO33 (Diabexa-2) induces significant exon 2 skipping of the PTPN1 transcript, and therefore this AO may potentially induce a decrease in functional PTP1B protein production.

[0205] Western blotting was performed to evaluate the effects of the 2'OMePS and PMO forms of PTPN1 1E2A(+5+29) on the inhibition of PTP1B protein, compared with untreated samples. Seventy-two hours after transfection, IHH cells were harvested and stored in a -80°C freezer. Frozen transfected IHH cell pellets were thawed and homogenized in SDS lysis buffer (0.5 M Tris-HCl pH 6.8, 3% SDS (w / v), and 10% glycerol (v / v)) containing protease inhibitors (Sigma). The homogenates were then centrifuged at 14,000 g for 3 minutes, after which the supernatants were removed and the protein concentrations of the supernatants estimated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins from the samples were then separated on nitrocellulose membranes (Biorad). The membrane was incubated with primary anti-PTP1B antibody (1:1000) (Cat. 5311S, Cell Signaling Technology) in 5% skim milk in TBS-T overnight at 4°C on a seesaw shaker. The membrane was then washed with TBS-T for 1 hour at room temperature on a shaker. After washing, the membrane was incubated with secondary anti-rabbit HRP antibody (1:10000) (Cat. 31460, Thermo Fisher Scientific) on a seesaw shaker for 1 hour at room temperature, followed by washing with TBS-T. Protein bands were visualized using a chemiluminescence-based procedure using the Clarity Western ECL detection kit according to the manufacturer's instructions (Biorad). Western blotting results showed that PTPN1 1E2A(+5+29)(AO33) treatment significantly reduced the expression level of PTP1B protein (Figure 16). Specifically, 400 nanomolar concentrations of the 2'OMePS form of AO33 (Diabexa-2) induced a 31% reduction or inhibition of PTP1B protein, and 7.5 μM and 15 μM concentrations of the PMO form of AO33 (Diabexa-2) induced a 20% and 50% reduction or inhibition of PTP1B protein (Figure 16).

[0206] AO1, 31-36 showed excellent human PTPN1 exon-2 skipping, resulting in the induction of a premature stop codon in exon-3, leading to a significant reduction in the expression level of functional PTPN1 gene product. Furthermore, Western blotting results demonstrated that AO33 (Diabexa-2):PTPN1 1E2A(+5+29) treatment significantly reduced the expression level of PTP1B protein.

[0207] Example 5. PTP1B Expression in Cancer Cells PTP1B expression was analyzed in various cancer cell lines, including breast cancer (MCF-7, MDA), mesothelioma (JU77, One58), glioblastoma (U87, U251), neuroblastoma (SH-SY5Y), medulloblastoma (DAOY), and liver cancer (HepG2), using a primer pair (PTP1B_Ex1F: 5′-GTG ATG CGT AGT TCC GGC TG-3′, PTP1B_Ex6R: 5′-CAG GGA CTC CAA AGT CAG GC-3′) that gave a 730-bp product (Figure 20). Briefly, the conditions were 55°C for 30 minutes, 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 1 minute, and 68°C for 2 minutes. PCR products were then separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized with a Fusion Fx gel documentation system.

[0208] Example 6: Synthesis and Characterization of Thiomorpholino Oligonucleotides (TMOs) As previously mentioned, the TMO chemistry may address issues commonly associated with ASO efficacy. Unlike PMOs, which are currently used in various approved splice-switching ASO drugs, TMOs can be efficiently synthesized using solid-phase oligonucleotide synthesis methods. This reduces the cost of synthesis work and, consequently, the cost of treatment. Utilizing solid-phase synthesis also means that TMOs can be synthesized as large-scale therapeutics. Furthermore, TMOs have been shown to have superior exon-skipping activity at lower concentrations, e.g., 5 and 10 nM, than other known chemistries such as 2′-O-methyl (2′-OMe) and 2′-O-methoxyethyl (2′-MOE) oligonucleotides [9]. This results in minimal dosages that may improve drug safety profiles. Therefore, TMOs represent a new and promising class of nucleic acid analogs for the development of oligonucleotide therapeutics.

[0209] We developed thiomorpholino (TMO) antisense oligonucleotides (ASOs) to induce exon 2 skipping in the PTPN1 pre-mRNA transcript, thereby eliminating PTP1B protein expression. To this end, various exon-skipping ASOs were designed, synthesized, and evaluated in various in vitro model systems (Table 4). [Table 4-1] [Table 4-2]

[0210] The TMO ASO was synthesized using the procedures described herein. All other ASOs were purchased from Syngenis (Bentley, WA, Australia). The PMO ASO was purchased from Gene Tools (Philomas, OR, USA). Commercial reagents and solvents were purchased from Glen Research (Sterling, VA). All syntheses were performed on a 1 μmol scale using a 5′-O-dimethoxytritylnucleoside-5′-hemisuccinate-modified LCAA-CPG (500 Å) solid support purchased from Glen Research (Sterling, VA).

[0211] Morpholino monomer: adenosine-(N-bz)-methyleneoxy-DMTr-morpholino-cyanoethyl-N,N-diisopropyl-phosphordiamidite (mA Bz ), guanosine-(N2-ib)-methyleneoxy-DMTr-morpholino-cyanoethyl-N,N-diisopropyl-phosphordiamidite (mG iBu ), cytidine-(N-bz)-methyleneoxy-DMTr-Mrpholino-cyanoethyl-N,N-diisopropyl-phosphordiamidite (mC Bz ), thymidine-methyleneoxy-DMTr-orino-cyanoethyl-N,N-diisopropyl-phosphordiamidite (mT) was purchased from ChemGenes Corporation (Wilmington, MA).

[0212] All solid-phase syntheses were performed using DMT ON. For the synthesis of TMO and TMO / DNA chimeras, 0.1 M of appropriately protected morpholino 3'-phosphorodiamidites and appropriately protected 2'-deoxyribonucleoside 3'-phosphoramidites were dissolved in anhydrous acetonitrile, and detritylation was achieved using a 3% solution of trichloroacetic acid in dichloromethane. A 50-second coupling time was used for the condensation reaction. Condensation was followed by sulfurization and capping. After synthesis was complete, the resin was treated with concentrated ammonia solution at 55°C for 16 hours, which cleaved the oligonucleotides from the CPG and removed the protecting groups on the oligonucleotides.

[0213] Solid-phase synthesis of TMO / DNA chimeras and TMOs DMTr-ON TMOs were synthesized by the phosphoramidite method on an ABI394 synthesizer (see below). The first reaction step of the synthesis cycle was 5'-dimethoxytrityl (DMT) deprotection of the 2'-deoxyribonucleoside monomer linked to a controlled pore glass solid support (CPG-500 support, Glen Research) using 3% trichloroacetic acid in dichloromethane. The second reaction step involved condensation of the resulting CPG-linked 5'-hydroxyl-2'-deoxyribonucleoside with a 6'-DMT-morpholino nucleoside 3'-phosphordiamidite to give mA. Bz ,mG iBu ,mC Bz The synthesis of the mT phosphoramidite and commercially available 5'-DMT-2'-deoxyribonucleoside 3'-phosphoramidite was achieved in anhydrous acetonitrile containing 0.2 M 5-ethylthio-1H-tetrazole (ETT) as the activating agent (condensation time: 50 s). The third reaction step was sulfurization using 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) as the sulfurizing reagent. In the final step, unreacted hydroxyl groups were acylated using capping reagents (Cap A: tetrahydrofuran / acetic anhydride and Cap B: 16% 1-methylimidazole in tetrahydrofuran). The synthesis cycle was then repeated until the TMO was prepared. [Table A]

[0214] After synthesis was completed, cleavage of these 5'-protected DMT-on oligonucleotides from the solid support and deprotection of the base and phosphorus protecting groups were carried out in 0.5 ml of 28% aqueous ammonia at 55 °C for 16 hours. Subsequent filtration to remove CPG from the oligonucleotides was performed through a microspin centrifuge filter with a 0.2 μm pore size. The resulting filtrate was evaporated to dryness using a SpeedVac (Thermo Fisher Scientific). The resulting residue was redissolved in 0.75 ml of a 3% acetonitrile / water mixture and filtered through a microspin centrifuge filter to obtain the total reaction product mixture. A small amount of this sample (5 μl diluted with 20 μl of HPLC water) was removed and analyzed by LC-MS chromatography. The remaining portion of this solution, along with the reaction mixture, was purified by RP-HPLC column chromatography. Fractions containing the pure compound were combined, evaporated to dryness, and subjected to LC-MS analysis.

[0215] The pure DMT-ON sample was dissolved in 0.5 ml of the detritylation mixture. After detritylation was complete, the mixture was neutralized with 5 μl of triethylamine. The sample was then filtered using a microspin centrifugal filter, and the filtrate containing the sample was purified by RP-HPLC column chromatography. The fractions containing the final DMT-OFF product were combined and evaporated to dryness in a SpeedVac. The purity of the product was determined by LC-MS analysis of the concentration of the sample in nanodroplets before storing the sample at -20 °C. The TMO was characterized by LC-MS analysis, and the results are shown in Table 5.

[0216] After synthesis was completed, cleavage of these 5'-protected DMT-on oligonucleotides from the solid support and deprotection of the base and phosphorus protecting groups were carried out in 0.5 ml of 28% aqueous ammonia at 55 °C for 16 hours. Subsequent filtration to remove CPG from the oligonucleotides was performed through a microspin centrifuge filter with a 0.2 μm pore size. The resulting filtrate was evaporated to dryness using a SpeedVac (Thermo Fisher Scientific). The resulting residue was redissolved in 0.75 ml of a 3% acetonitrile / water mixture and filtered through a microspin centrifuge filter to obtain the total reaction product mixture. A small amount of this sample (5 μl diluted with 20 μl of HPLC water) was removed and analyzed by LC-MS chromatography. The remaining portion of this solution, along with the reaction mixture, was purified by RP-HPLC column chromatography. Fractions containing the pure compound were combined, evaporated to dryness, and subjected to LC-MS analysis.

[0217] The pure DMT-ON sample was dissolved in 0.5 ml of the detritylation mixture. After detritylation was complete, the mixture was neutralized with 5 μl of triethylamine. The sample was then filtered using a microspin centrifugal filter, and the filtrate containing the sample was purified by RP-HPLC column chromatography. The fractions containing the final DMT-OFF product were combined and evaporated to dryness in a SpeedVac. The purity of the product was determined by LC-MS analysis of the concentration of the sample in nanodroplets before storing the sample at -20 °C. The TMO was characterized by LC-MS analysis, and the results are shown in Table 5. [Table 5]

[0218] Cell culture and transfection of ASOs into cells Human hepatocellular carcinoma cell lines, including HepG2 (catalog no. 85011430) and Huh-7 (catalog no. JCRB0403), and the human pancreatic cancer cell line PANC-1 (catalog no. 87092802), were obtained from Cell Bank Australia. The immortalized human hepatocyte (IHH) and mouse hepatocyte (AML-12) cell lines were provided by Professor Grant Ramm (Head of the Liver Fibrosis Group, Liver Unit, QIMR Berghofer). The human triple-negative breast cancer cell line MDA-MB231 was provided by Associate Professor Stacy Edwards (Director of the Institute for Functional Cancer Genomics, QIMR Berghofer).

[0219] The HepG2 cell line was cultured in 10% FBS Eagle's Minimum Essential Medium (ATCC). Both the 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). The Panc-1 and Huh-7 cell lines were cultured in 10% FBS Dulbecco's Modified Eagle's Medium. All cell lines were cultured in a humidified cell chamber at 37°C and 5% CO2. Cells were cultured to reach 70-90% confluency, and then 24 hours before transfection, 2.5-4 × 10 cells were added. 4 Cells were seeded into 24-well plates (Thermo Fisher Scientific) at a density of 1000 (cells / mL). The next day, ASOs were transfected at the desired concentration using Lipofectamine RNAiMAX Reagent according to the original manufacturer's protocol. 24 hours after transfection, cells were harvested for RNA extraction and RT-PCR analysis unless otherwise noted.

[0220] RNA extraction and RT-PCR RNA was extracted from transfected cells using the ISOLATE II RNA Mini Kit (Bioline, Eveleigh, NSW, Australia) according to the manufacturer's instructions. Human PTPN1 exon 2 skipping products (product size: 639 bp) and non-skipping products (product size: 730 bp), and mouse Ptpn1 exon 2 skipping products (product size: 493 bp) and non-skipping products (product size: 584 bp) were isolated using a SuperScript® III One-Step RT-PCR Kit (Thermo Fisher Scientific) with the human PTPN1 primer pair (PTP1B_Ex1F: 5′-GTG ATG CGT AGT TCC GGC TG-3′, PTP1B_Ex6R: 5′-CAG GGA CTC CAA AGT CAG GC-3′) or the mouse Ptpn1 primer pair (Ptpn1_B_Ex1F: 5′-AGA TCG ACA AGG CTG GGA AC-3′, Ptpn1_B_Ex6R: 5′-TGA GCC TGA CTC TCG GAC TT-3′). PCR products were amplified using a PCR product library (Scientific). Briefly, 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. PCR products were then separated on a 2% agarose gel in Tris-acetate-EDTA buffer and visualized using a Fusion Fx gel documentation system. Densitometry was performed using ImageJ software.

[0221] Western blot analysis Western blot analysis was performed to evaluate the inhibitory effect of the lead TMO ASO on PTP1 B protein. Briefly, the ASO was transfected into IHH cells at 400 nM. 72 hours after transfection, the cells were harvested, and the cell pellet was homogenized in SOS lysis buffer (0.5 M Tris-HCl pH 6.8, 3% SOS (w / v), and 10% glycerol (v / v)) containing protease inhibitors (Sigma). The homogenate was then centrifuged at 14,000 g for 3 minutes, the supernatant was removed, and the protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). The protein samples were then run on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Biorad). The membrane was incubated with primary anti-PTP1B antibody (1:1000) (Cell Signaling Technology, catalog 5311S) in 5% skim milk in TBS-T overnight at 4°C on a seesaw shaker, then washed with TBS-T for 1 hour at room temperature on a shaker, followed by incubation with secondary anti-rabbit HRP antibody (1:10000) (Cat. 31460, Thermo Fisher Scientific) for 1 hour at room temperature and washing three times with TBS-T. Protein bands were visualized using a chemiluminescence-based procedure using the Clarity Western EGL detection kit on a Fusion Fx gel documentation system according to the manufacturer's instructions (Biorad).

[0222] Example 7: Thiomorpholino antisense oligonucleotides (TMOs) targeting human or mouse PTPN1 pre-mRNA transcripts Initial screening experiment of TMO ASO Thiomorpholino (TMO, Figure 21) antisense oligonucleotides (ASOs) were developed to inhibit human PTP1B protein expression. TMO ASOs were synthesized using the procedure described by Langner et al. [10, 11]. The sequences of all TMO ASOs listed in this example are shown in Table 4. The ASOs bind to the human PTPN1 pre-mRNA transcript and induce exon 2 skipping in the transcript, thereby abolishing human PTP1B protein expression. Exon-skipping ASOs were designed and evaluated (Table 4). Briefly, ASOs (50–400 nM) were transfected into either hepatocellular carcinoma (HepG2) cells or immortalized human hepatocyte (IHH) cells as previously described. After 24 h of incubation, cells were harvested, and RNA was extracted and then subjected to RT-PCR.

[0223] The results demonstrated that ASOs 2–4 (a mixture of TMO, 2′-O-methyl RNA, and DNA) induced human PTPN1 exon 2 skipping in HepG2 cells at different levels (Figure 30). Notably, although ASO4 is 7 nucleotides shorter than ASOs 2 and 3, the results showed that approximately 42% of exon 2 skipping in the PTPN1 transcript was observed at 400 nM compared to untreated cells (Figure 30, densitometry).

[0224] Comparison of the chemical structures of TMO, 2′-OMePS, and 2′-MOEPS Inspired by these results, we continued synthesizing ASOs 5–7 and evaluated them in IHH cells. This cell line represents normal hepatocytes and is more relevant to the conditions of type 2 diabetes and obesity. The newly synthesized ASOs share the exact same sequence but are synthesized with different chemical structures. ASO5 is a complete TMO ASO (except for the 3′-terminal DNA nucleotide), ASO6 is a complete 2′-O-methyl RNA, and ASO7 is a complete 2′-O-methoxyethyl RNA. The results showed that all three ASOs efficiently induced exon 2 skipping. However, ASO5 performed significantly better than the other two ASOs at all concentrations (Figure 31). Notably, at 50 nM, ASO5 induced nearly twice the amount of exon skipping product (639 bp) of ASO6 (50% compared to 27% for ASO6) and was comparable to ASO7 at 100 nM (51% compared to 50% for ASO7). At 100 nM, ASO5 continued to drive exon skipping efficacy by resulting in 62% skipped product compared to 45% and 51% for ASO6 and 7, respectively. This trend continued at 200 nM and 400 nM concentrations, where ASO5 induced 75 and 80% exon skipping compared to 55 and 66% for ASO6 and 69 and 78% for ASO7 (Figure 31).

[0225] ASO5 Microwalking To fine-tune the ASO design, we used microwalking techniques to engineer six additional ASOs (8–13) that shift one to three nucleotides toward either the 5′ or 3′ end of the transcript. The ASOs were evaluated in IHH cells as described above. Results showed that all ASOs induced efficient exon skipping at all concentrations (Figure 32). Notably, ASO5 remained the overall best ASO for exon 2 skipping efficacy (50, 62, 75, and 80% skipping induced at 50 nM–400 nM, respectively). Among the ASOs microwalked toward the 5′ end of the PTPN1 transcript, ASO9 performed best (51, 61, 71, and 84% skipping induced at 50 nM–400 nM, respectively), followed by ASO10 and ASO8 (Figure 32).

[0226] On the other hand, when microwalking towards the 3′ end of the PTPN1 transcript, ASO13 and 11 showed comparable efficacy by inducing exon 2 skipping ranging from 43 to 79% for ASO13 and 45 to 80% for ASO11, whereas ASO12 was less efficient compared to the other ASOs (Figure 33).

[0227] Analysis of PTP1B protein expression in normal human hepatocytes after treatment with TMO ASO We selected the three best candidates, ASO5, 9, and 11, and performed Western blots to evaluate the efficacy of the ASOs at the protein level. Briefly, IHH cells were transfected with 400 nM ASOs for 72 hours and then harvested for both RNA and protein assays. At the RNA level, ASO33 (25-mer full 2'-OMePS) as a positive control induced the greatest amount of exon 2 skipping in the PTPN1 transcript (81%, Figure 34A and B). Surprisingly, among the TMO ASOs, ASO9 performed better than ASO5, which was not the case previously. On the other hand, ASO11 produced a lower amount of exon skipping product than the other two TMOs (Figure 34A and B). Notably, at the protein level, ASO5 achieved 91% PTP1B protein inhibition, better than the other ASOs tested in this study (Figure 34C and D). ASOs 9 and 11 continued to perform well in this study, achieving 82 and 77% protein inhibition, respectively.

[0228] Evaluation of the 25-mer TMO compared to the lead 18-mer TMO ASO Furthermore, we also synthesized a 25-mer TMO (ASO1) using the corresponding target coordinates of (+5+29) in the PTPN1 transcript and tested it against the best ASO candidates, ASO5, 9, and 11. The results showed that ASO1 performed significantly better than the other three 18-mer ASOs. This is not surprising, as longer ASOs have better binding affinity to RNA targets. Notably, at 50 nM, ASO1 induced 60% exon skipping, nearly twice the skipping efficacy of ASO5 (38%) and 1.5 times that of ASO9 (43%) and ASO11 (41%) (Figure 35). This trend continued at 100 nM and 200 nM, with ASO1 yielding 78% and 90% skipped products, respectively, 1.5-fold higher than the other three ASOs, which yielded 53 and 62% (ASO5), 59 and 63% (ASO9), and 55 and 64% (ASO11). At 400 nM, ASO1 continued to excel in skipping efficiency, inducing 94% exon-skipped products compared with 71, 80, and 72% for ASO5, 9, and 11, respectively (Figure 35).

[0229] Evaluation of exon skipping efficacy of lead TMO ASO in hepatocellular carcinoma cells To test the lead TMO ASOs in liver cancer cells, ASOs 5, 9, and 11 were transfected at concentrations of 50 nM–400 nM into Huh-7 cells (4 × 10 cells) 24 h before transfection. 4The cells were transfected into normal human hepatocytes (seeded at 1000 cells / well). The cells were then incubated for 24 hours and then harvested for RNA extraction and RT-PCR. The results demonstrated that all three ASOs efficiently induced exon 2 skipping in the PTPN1 transcript (Figure 36). Notably, unlike the case of normal human hepatocytes, where ASO5 was the best ASO, ASO9 and 11 performed better than ASO5 at all concentrations except for ASO9 at 100 nM. Densitometry analysis showed that ASO9 produced the highest amount of exon 2 skipping product (49–83%, respectively, from 50–400 nM), followed by ASO11 (46–75%, respectively, from 50–400 nM). ASO5 did not perform well in this case, as it only induced 38–73% exon skipping (Figure 36).

[0230] Evaluation of exon-skipping efficacy of lead TMO ASO in human pancreatic cancer cells To further evaluate the effects of PTP1B protein inhibition in cancer, we tested the lead ASO against the human breast ductal adenocarcinoma cell line PANC-1. Briefly, ASOs were transfected into cells (4 x 10 cells) at concentrations of 50 nM to 400 nM as described above, 24 h before transfection. 4 Cells were transfected into PTPN1 cells (seeded at 1000 cells / well). After analysis, the results showed that all three ASOs efficiently induced exon 2 skipping in the PTPN1 transcript (Figure 37). Surprisingly, in this case, ASO11 produced the highest amount of exon 2 skipping product at 400 nM (74%, Figure 37), followed by ASO9 (69%) and ASO5 (59%). Interestingly, at 200 nM, all three ASOs functioned similarly, with ASO5 and ASO9 inducing 59% exon skipping and ASO11 inducing 57% skipping. From 50 to 100 nM, ASO9 and 11 functioned comparably, while ASO5 was less efficient.

[0231] Evaluation of exon-skipping efficacy of lead TMO ASO in human triple-negative breast cancer cells To evaluate the effect of PTP1B inhibition in the triple-negative breast cancer cell line MDA-MB231, we inoculated the lead ASO into cells (4 × 10 cells) at concentrations between 50 nM and 400 nM 24 h before transfection. 4 The three ASOs were tested by transfecting cells (seeded at 1000 cells / well). The results showed that all three ASOs induced exon 2 skipping in the PTPN1 transcript very efficiently (Figure 38). Interestingly, in this case, ASO5 was the best ASO for inducing exon skipping, unlike in liver cancer and pancreatic cancer. At 50 and 100 nM, ASO5 produced 79 and 88% exon 2 skipped product, respectively, followed by ASO9 (77 and 86%) and ASO11 (71 and 80%, Figure 38). At 200 nM, all three ASOs, performing similarly to ASO9 and 11, induced 94% exon skipping, while ASO5 induced a slightly lower amount of skipped product at 93%. Notably, at 400 nM, ASO5 maintained 93% efficacy, while ASO9 and 11 decreased to 84 and 87%, respectively.

[0232] Example 8: Thiomorpholino antisense oligonucleotides targeting mouse Ptpn1 pre-mRNA transcripts Initial screening of mouse TMO ASO To test the TMO ASOs in vivo in a mouse model, we designed and synthesized several mouse ASOs targeting similar coordinates in the Ptpn1 gene as the three best TMOs in humans (ASOs 17–19, Table 4). Briefly, ASOs 17–19 (50–400 nM) were transfected into mouse hepatocyte AML-12 cells using Lipofectamine RNAiMax. After incubating the cells for 24 hours, RNA was harvested and extracted for RT-PCR. The results demonstrated that all three ASOs induced exon 2 skipping in the mouse Ptpn1 transcript at different levels (Figure 39). Surprisingly, ASO18 performed better than the other two ASOs at all concentrations, achieving 41% exon 2 skipping at 400 nM (Figure 39).

[0233] Further investigation of mouse versus human PTPN1 transcripts and evaluation of newly designed ASOs To improve mouse ASO efficacy, we investigated the splicing map of the mouse Ptpn1 transcript and compared it with that of humans. We found that the splicing factors and branch points in this region in mice differ from those in humans (Figure 4). To address this issue, we designed three additional ASOs (20-22) (Table 4) and synthesized them with the 2'-MOEPS chemistry. These ASOs targeted mouse splicing factor binding sites and were tested using the same strategy as described above.

[0234] The results demonstrated that only ASO21 improved the exon skipping induction performance (Figure 41). The exon skipping rate was recorded as 30–57% in a dose-dependent manner (50–400 nM, respectively, Figure 41). On the other hand, ASO20 induced a smaller amount of exon skipping product (8–15% at 50–400 nM, respectively), while ASO22 induced negligible exon skipping.

[0235] Evaluation of 25-mer vs. 18-mer mouse ASOs in 2'-MOE chemistry To investigate whether ASO length affects exon skipping efficacy, we designed and synthesized a 25-mer mouse ASO23 using a 2′-MOE chemistry based on a human positive control sequence (ASO33) and tested it against the best 18-mer mouse 2′-MOE ASO21. Briefly, the ASOs were transfected into AML-12 cells at 50–400 nM and collected for further analysis. Results showed that ASO23 significantly improved exon skipping performance at all concentrations (Figure 42). Exon-skipped products were generated in a dose-dependent manner for both ASOs, with the 25-mer generating 46–77% exon-skipped products (50–400 nM, respectively; Figure 42). On the other hand, the 18-mer ASO generated only 26–58% exon-skipped products (50–400 nM, respectively).

[0236] Comparison of 25-mer TMO ASO with 25-mer MOE ASO. Having tested the 25-mer 2'-MOE ASO23 and observed excellent data, we attempted to synthesize a 25-mer TMO variant of the same target coordinates (ASO24) and tested it against ASO23. The ASO was subjected to the same testing protocol as described in the previous experiment. Surprisingly, the results showed that ASO24 was significantly less efficient than ASO23 (Figure 43A). Densitometry analysis demonstrated that TMO induced lower amounts of exon skipping products (9-31% at 50-400 nM, respectively) than 2'-MOE (26-67% at 50-400 nM, respectively, Figure 43A).

[0237] TMO ASOs are known to be highly efficient at low doses. Therefore, we repeated the same experiment with ASO24 and 23 described above, but at a concentration range of 5-50 nM. Interestingly, both ASOs performed significantly better in this concentration range compared to higher doses (Figure 43B). At 5 nM-25 nM, ASO24 was less efficient than ASO23, yielding 55, 70, and 83% exon skipping products compared to 78, 93, and 94% obtained by ASO23. Notably, at 50 nM, ASO24 performed comparable to ASO23, with an exon skipping rate of 89% compared to 94% induced by ASO23 (Figure 43B).

[0238] References 1. McDonald CM, Wong B, Flanigan KM, et al. Placebo-controlled Phase 2 Trial of Drisapersen for Duchenne Muscular Dystrophy. Ann Clin Transl Neurol. 2018;5(8):913-926. 2.Dirin M, Winkler J.Influence of diverse chemical modifications on the ADME characteristics and toxicology of antisense oligonucleotides.Expert Opin Biol Ther.2013;13(6):875-888. 3. Yip SC, Saha S, Chernoff J. PTP1B: a double agent in metabolism and oncogenesis.Trends Biochem Sci.2010;35(8):442-449. 4.Dube N,Tremblay ML.Beyond the metabolic function of PTP1B.Cell Cycle.2004;3(5):550-553. 5.Tautz L.PTP1B: a new therapeutic target for Rett syndrome.J Clin Invest.2015;125(8):2931-2934. 6.Krishnan N,Krishnan K,Connors CR,et al.PTP1B inhibition suggests a therapeutic strategy for Rett syndrome.J Clin Invest.2015;125(8):3163-3177. 7.Xu Q,Wu N,Li X.et al.Inhibition of PTP1B blocks pancreatic cancer progression by targeting the PKM2 / AMPK / mTOC1 pathway.Cell Death Dis.2019;10:874. 8.Tai WT,Chen YL,Chu PY,et al.Protein tyrosine phosphatase 1B dephosphorylates PITX1 and regulates p120RasGAP in hepatocellular carcinoma [published correction appears in Hepatology.2017 Jun;65(6):2135-2136].Hepatology.2016;63(5):1528-1543. 9.Le BT,Paul S,Jastrzebska K,Langer H,Caruthers MH,Veedu RN.Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing.Proc Natl Acad Sci U S A.2022;119(36):e2207956119. 10.Langner HK,Jastrzebska K,Caruthers MH.Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras.J Am Chem Soc.2020;142(38):16240-16253. 11.Paul S.,Caruthers M.,Synthesis of Backbone Modified Morpholino Oligonucleotides and Chimeras Using Phosphoramidite Chemistry,US Patent 11,230,565 B2,2022.

Claims

1. 1. An isolated or purified antisense oligomer targeted to a nucleic acid molecule encoding human protein tyrosine phosphatase non-receptor type 1 (PTPN1) pre-mRNA, said antisense oligomer having a nucleobase sequence selected from SEQ ID NOs: 42-57, 69-73, or 75, and having a modified backbone structure, wherein said antisense oligomer inhibits expression of PTP1B.

2. 2. The antisense oligomer of claim 1, wherein the antisense oligomer induces alternative splicing of human PTPN1 pre-mRNA via exon skipping.

3. The antisense oligomer of claim 1 or 2, wherein the antisense oligomer induces exon skipping of exon 2 in human PTPN1 pre-mRNA.

4. 4. The antisense oligomer of any one of claims 1 to 3, wherein the antisense oligomer comprises at least one nucleotide position to which an alternative chemical structure or modification selected from (i) a modified sugar moiety, (ii) resistance to RNase H, and / or (iii) an oligomer-mimetic chemical structure is applied, and / or the antisense oligomer is modified by (i) a chemical conjugate coupled to a moiety by a linker, the chemical conjugate being selected from vitamin E, cholesterol, and / or N-acetylgalactosamine (GalNAc), (ii) tagging with a cell-penetrating peptide, and / or (iii) encapsulation in a liposomal nanoparticle structure.

5. 5. The antisense oligomer of any one of claims 1 to 4, having a nucleobase sequence selected from SEQ ID NOs: 55-56, 57A, and 69-70.

6. The antisense oligomer of any one of claims 1 to 5, wherein, if uracil is present in the antisense oligomer, the uracil (U) of the antisense oligomer is substituted with thymine (T).

7. The antisense oligomer according to any one of claims 1 to 6, wherein the antisense oligomer is a thiomorpholino oligomer (TMO).

8. The antisense oligomer according to any one of claims 1 to 7, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) or a 2'-O-methoxyethyl RNA (2'-O-MOE).

9. The antisense oligomer according to any one of claims 1 to 8, wherein the antisense oligomer is a 2'-O-methyl RNA oligomer (2'-OMe).

10. The antisense oligomer of claim 1, wherein the antisense oligomer comprises SEQ ID NO: 42-57 or 69-73 or 75.

11. The antisense oligomer of claim 1 , wherein the antisense oligomer comprises SEQ ID NO:

42.

12. The antisense oligomer of claim 1, wherein the antisense oligomer comprises SEQ ID NO:

46.

13. The antisense oligomer of claim 1 , wherein the antisense oligomer comprises SEQ ID NO:

50.

14. The antisense oligomer of claim 1, wherein the antisense oligomer comprises SEQ ID NO:

52.

15. 15. A method for inducing alternative splicing of PTPN1 pre-mRNA, the method comprising the step of providing one or more antisense oligomers according to any one of claims 1 to 14 and allowing the oligomer(s) to bind to a target nucleic acid site.

16. 1. A pharmaceutical composition for treating, preventing, or alleviating the effects of a PTP1B-related disease in a subject, comprising: One or more antisense oligomers according to any one of claims 1 to 14; one or more pharmaceutically acceptable carriers and / or diluents; Including, composition.

17. 17. The pharmaceutical composition of claim 16, wherein the disease associated with PTP1B is type 2 diabetes or obesity.

18. The pharmaceutical composition of claim 16, wherein the disease associated with PTP1B is Rett syndrome.

19. 17. The pharmaceutical composition of claim 16, wherein the PTP1B-associated disease is cancer, including breast cancer, pancreatic cancer, liver cancer, colon cancer, gastric cancer, and melanoma.

20. A method for treating, preventing or alleviating the effects of a disease associated with PTP1B, comprising administering to a subject in need thereof an effective amount of one or more antisense oligomers or a pharmaceutical composition comprising one or more antisense oligomers according to any one of claims 1 to 14.

21. 21. The method of claim 20, wherein the PTP1B-related disease is type 2 diabetes or obesity.

22. 21. The method of claim 20, wherein the disease associated with PTP1B is Rett syndrome.

23. 21. The method of claim 20, wherein the PTP1B-associated disease is cancer, including breast cancer, pancreatic cancer, liver cancer, colon cancer, gastric cancer, and melanoma.

24. An expression vector comprising the antisense oligomer according to any one of claims 1 to 14.

25. 15. Use of the purified and isolated antisense oligomer of any one of claims 1 to 14 for the manufacture of a medicament for treating, preventing or ameliorating the effects of a disease associated with PTP1B.

26. 15. Use of the purified and isolated antisense oligomer of any one of claims 1 to 14 for treating, preventing or ameliorating the effects of a disease associated with PTP1B.

27. The use according to claim 19 or 20, wherein the disease associated with PTP1B is type 2 diabetes and / or obesity.

28. The use according to claim 19 or 20, wherein the disease associated with PTP1B is Rett syndrome.

29. The use according to claim 19 or 20, wherein the disease associated with PTP1B is cancer, including breast cancer, pancreatic cancer, liver cancer, colon cancer, gastric cancer and melanoma.

30. A kit for treating, preventing or alleviating the effects of a PTP1B-related disease in a subject, comprising at least an antisense oligomer described in any one of claims 1 to 14 packaged in a suitable container together with instructions for use thereof.

31. The kit of claim 30, wherein the PTP1B-related disease is type 2 diabetes and / or obesity.

32. The kit of claim 30, wherein the disease associated with PTP1B is Rett syndrome.

33. The kit of claim 30, wherein the PTP1B-associated disease is cancer, including breast cancer, pancreatic cancer, liver cancer, colon cancer, gastric cancer, and melanoma.

34. 1. An isolated or purified antisense oligomer targeted to a nucleic acid molecule encoding mouse protein tyrosine phosphatase non-receptor type 1 (PTPN1) pre-mRNA, said antisense oligomer having a nucleobase sequence selected from SEQ ID NOs: 58-68 and having a modified backbone structure, wherein said antisense oligomer inhibits expression of mouse PTP1B.

35. 35. The antisense oligomer of claim 34, wherein the antisense oligomer induces alternative splicing of PTPN1 pre-mRNA via exon skipping.

36. the antisense oligomer is (i) a modified sugar moiety; (ii) resistance to RNase H, and / or (iii) oligomeric mimetic chemical structures; 36. The antisense oligomer of claim 34 or 35, comprising one or more nucleotide positions to which an alternative chemical structure or modification selected from:

37. the antisense oligomer is (i) a chemical conjugate coupled to a moiety by a linker, the chemical conjugate being selected from vitamin E, cholesterol, and / or N-acetylgalactosamine (GalNAc); (ii) tagging with a cell-penetrating peptide, and / or (iii) being encapsulated in a liposomal nanoparticle structure; 37. The antisense oligomer of any one of claims 34 to 36, further modified by:

38. 38. The antisense oligomer of any one of claims 34 to 37, wherein, if uracil is present in the antisense oligomer, the uracil (U) of the antisense oligomer is substituted with thymine (T).

39. The antisense oligomer according to any one of claims 34 to 38, wherein the antisense oligomer is a thiomorpholino oligomer (TMO).

40. The antisense oligomer according to any one of claims 34 to 39, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) or a 2'-O-methoxyethyl RNA (2'-O-MOE).

41. The antisense oligomer according to any one of claims 34 to 40, wherein the antisense oligomer is a 2'-O-methyl RNA oligomer (2'-OMe).

42. 1. An isolated or purified antisense oligomer targeted to a nucleic acid molecule encoding simian protein tyrosine phosphatase non-receptor type 1 (PTPN1) pre-mRNA, said antisense oligomer having a nucleobase sequence selected from SEQ ID NOs: 76-81 and having a modified backbone structure, wherein said antisense oligomer inhibits expression of simian PTP1B.

43. 43. The antisense oligomer of claim 42, wherein the antisense oligomer induces alternative splicing of PTPN1 pre-mRNA via exon skipping.

44. the antisense oligomer is (i) a modified sugar moiety; (ii) resistance to RNase H, and / or (iii) oligomeric mimetic chemical structures; 44. The antisense oligomer of claim 42 or 43, comprising at least one nucleotide position to which an alternative chemical structure or modification selected from:

45. the antisense oligomer is (i) a chemical conjugate coupled to a moiety by a linker, the chemical conjugate being selected from vitamin E, cholesterol, and / or N-acetylgalactosamine (GalNAc); (ii) tagging with a cell-penetrating peptide, and / or (iii) being encapsulated in a liposomal nanoparticle structure; 45. The antisense oligomer of any one of claims 42 to 44, further modified by:

46. 46. ​​The antisense oligomer of any one of claims 42 to 45, wherein, if uracil is present in the antisense oligomer, the uracil (U) of the antisense oligomer is substituted with thymine (T).

47. The antisense oligomer according to any one of claims 42 to 46, wherein the antisense oligomer is a thiomorpholino oligomer (TMO).

48. The antisense oligomer according to any one of claims 42 to 47, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) or a 2'-O-methoxyethyl RNA (2'-O-MOE).

49. The antisense oligomer of any one of claims 42 to 48, wherein the antisense oligomer is a 2'-O-methyl RNA oligomer (2'-OMe).

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