Compositions and methods for delivery of truncated midkine proteins

Abstract

The present disclosure relates generally to novel compositions and methods for the delivery of truncated midkine proteins, and / or polynucleotides encoding such truncated human midkine proteins, in which the amino acid sequence encoded by exon 4 of human midkine is absent or partially absent.

Description

[Technical Field]

[0001] Related Applications This application claims priority from Australian Patent Application No. 2022 / 902231 entitled "Compositions and Methods for Delivery of Truncated Midkine Proteins", filed on August 9, 2022, and Australian Patent Application No. 2023 / 901841 entitled "Compositions and Methods for Delivery of Truncated Midkine Proteins", filed on June 9, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic form, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure generally relates to novel compositions and methods for the delivery of truncated midkine proteins. In particular, the disclosure relates to truncated human midkine proteins in which the amino acid sequence encoded by exon 4 of human midkine is absent or partially absent, and / or compositions comprising polynucleotides encoding such truncated human midkine proteins. The disclosure also relates to the use of these compositions for treating individuals having or susceptible to midkine-related diseases or disorders. [Background technology]

[0004] Midkine (MDK) is a heparin-binding growth factor found as the product of a gene transiently expressed during retinoic acid-induced differentiation of embryonic carcinoma (EC) cells. It is a 13-kDa polypeptide rich in basic amino acids and cysteine (Kadomatsu et al. (1988) Biochem. Biophys. Res. Commun., 151:1312-1318; Tomokura et al. (1999) J. Biol. Chem., 265:10765-10770; Muramatsu T (2014) Brit J Pharmacol 171:814-826).

[0005] Midkine is known to have various biological activities. For example, midkine expression is known to be increased in human cancer cells. This increased expression has been confirmed in various cancers, including esophageal cancer, thyroid cancer, bladder cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, mesothelioma, kidney cancer, head and neck cancer, melanoma, uterine / cervical cancer, ovarian cancer, osteosarcoma, chronic lymphocytic leukemia, and Wilms' tumor (Muramatsu (2002) J. Biochem. 132:359-371; Jones (2014) Brit J Pharm 171:2925-2939). Furthermore, midkine enhances cancer cell proliferation, survival, and migration, promotes angiogenesis, contributes to cancer progression and metastasis, and regulates the tumor immune microenvironment. Midkine is also a major determinant of response to cancer treatments, including chemotherapy and immunotherapy.

[0006] Midkine is also known to play a central role in regulating immune and inflammatory responses (Heradon G et al. (2019) Frontier Pharmacol 10:377, Aynacioglu AS et al. (2018) Modern Rheumatology 29:567-571, Sorrelle N et al. (2017) J Leukoc Biol 102:277-286). For example, midkine knockout mice lacking the midkine gene exhibit reduced vascular damage during ischemic injury and reduced neointima formation after nephritis. Furthermore, such knockout mice exhibit significantly reduced adhesion in rheumatoid arthritis models and after surgery (WO2000 / 10608, WO2004 / 078210). Midkine is known to be involved in inflammatory and autoimmune diseases such as arthritis (both rheumatoid and osteoarthritis), postoperative adhesions, inflammatory bowel disease, autoimmune myocarditis, chronic kidney disease, psoriasis, lupus, asthma, and multiple sclerosis associated with T regulatory cell dysfunction (Takeuchi H (2014) Brit J Pharmacol 171:931-935). Furthermore, midkine is known to promote the migration, activation, and functional orientation of inflammatory cells such as macrophages and neutrophils. Because the recruitment and harmful behavior of neutrophils and macrophages are necessary for the establishment of an inflammatory response in diseased tissues, deficiency or blockade of midkine action prevents inflammation-based diseases in animal models (WO 1999 / 03493). However, no midkine-based therapy has progressed beyond preclinical experimental testing. This limitation is exemplified by monoclonal antibodies targeting midkine, which have shown benefit only when administered in a prophylactic mode and have failed in a therapeutic mode once disease or tumors are established.

[0007] Thus, there remains a need for new compositions and methods with improved capabilities to regulate, inhibit, or reduce the abundance and activity of functional midkine. There is also a need for compositions with the ability to regulate, inhibit, or reduce the abundance and activity of functional midkine that are cost-effective and / or simple to manufacture.

[0008] Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification should not be taken as an admission that any or all of such matters form part of the basis of the prior art or were common general knowledge in the art to which this disclosure pertains as they existed prior to the priority date of each of the appended claims. Summary of the Invention

[0009] The present disclosure is based, inter alia, on the inventors' recognition that there exists a need for novel therapeutic strategies for targeting midkine (MDK) activity or function and treating conditions associated with midkine action. To this end, the inventors have developed novel compositions configured to deliver truncated forms of midkine protein, particularly truncated forms of midkine protein that are absent, partially absent, or substantially absent of the amino acid sequence encoded by exon 4 of human midkine. While not wishing to be bound by any one theory, the truncated forms of midkine protein of the present disclosure are believed to act as dominant-negative antagonists. The biologically active form of midkine is believed to be a homodimer comprising two full-length monomers. In this regard, the truncated forms of midkine protein of the present disclosure are non-functional but are believed to retain the ability to form heterodimers with full-length, functional midkine protein, thereby inhibiting the activity of full-length human midkine in cells or tissues. Alternatively, the truncated midkine protein may interfere with or compete with the ability of full-length midkine to interact with its ligands on the surface of or within the cell.

[0010] In efforts to produce truncated forms of the midkine protein of the present disclosure, the inventors developed midkine mRNAs that translate into forms of midkine protein lacking important functional domains, thereby reducing the biological activity of the full-length midkine protein. The presence of truncated forms of midkine protein was also confirmed by Western blot using polyclonal antibodies that recognize epitopes within regions (e.g., the N-terminal region) retained in the truncated midkine protein. Uniquely, these midkine mRNA and truncated midkine protein variants are distinguished from naturally occurring midkine splice variants because they involve only the removal of sequences from the C-terminal domain of midkine, leaving the N-terminal domain intact. Furthermore, the midkine mRNA and truncated midkine protein variants disclosed herein do not involve the addition of any amino acids or polynucleotide sequences unrelated to midkine.

[0011] The present inventors have surprisingly found that certain truncated variants are able to inhibit midkine function better than other variants, and in particular are able to inhibit midkine-mediated cell migration and / or reduce cancer cell survival.

[0012] Accordingly, the present disclosure provides a composition comprising: (i) a truncated human midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is absent, partially absent, or substantially absent, and / or (ii) A composition comprising a polynucleotide encoding the truncated human midkine protein in (i) is provided.

[0013] In one example, the composition comprises a truncated human midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is absent, partially absent, or substantially absent.

[0014] In one example, the amino acid sequence encoded by exon 4 of the human midkine protein is substantially absent from the truncated human midkine protein. The amino acid sequence encoded by exon 4 of human midkine is set forth in SEQ ID NO: 73. According to certain examples in which the amino acid sequence encoded by exon 4 of the human midkine protein is substantially absent, at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the sequence set forth in SEQ ID NO: 73 is absent from the truncated human midkine protein. In another example, the amino acid sequence encoded by exon 4 of the human midkine protein (i.e., the amino acid sequence set forth in SEQ ID NO: 73) is absent from the truncated human midkine protein.

[0015] In examples where the amino acid sequence encoded by exon 4 is substantially absent, one or more of the amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary may be present in the truncated midkine protein. For example, 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 83 may be present in the truncated human midkine protein. In some examples, the amino acid sequence set forth in SEQ ID NO: 83 is present in the truncated human midkine protein. Alternatively, or in addition, when the amino acid sequence encoded by exon 4 is substantially absent, one or more amino acids within exon 4 adjacent to the exon 4 / exon 5 boundary may be present. For example, 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 84 may be present in the truncated human midkine protein. In yet another example, one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary may also be present in a truncated midkine protein, and one or more amino acids within exon 4 adjacent to the exon 4 / exon 5 boundary may be present in a truncated human midkine protein. For example, 1, 2, 3, 4, or 5 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 83 may be present in a truncated human midkine protein, and 1, 2, 3, 4, or 5 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 84 may be present or substantially present in a truncated human midkine protein. In one example, the amino acid sequences set forth in SEQ ID NOs: 83 and 84 may be present in a truncated human midkine protein.

[0016] Alternatively, or in addition, one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary may be absent from the truncated midkine protein. For example, 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 85 may be absent or substantially absent from the truncated human midkine protein. In some examples, the amino acid sequence set forth in SEQ ID NO: 85 is absent from the truncated human midkine protein.

[0017] In one example, the truncated human midkine protein comprises an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 78. In another example, the truncated human midkine protein comprises the sequence set forth in SEQ ID NO: 78. In one example, the truncated human midkine protein consists of the sequence set forth in SEQ ID NO: 78.

[0018] In some examples, truncated human midkine proteins in which the amino acid sequence corresponding to exon 4 is absent or substantially absent are about 60 to about 80 amino acids in length. For example, the sequence of the truncated human midkine protein can be 60, or 61, or 62, or 63, or 64, or 65, or 66, or 67, or 68, or 69, or 70, or 71, or 72, or 73, or 74, or 75, or 76, or 77, or 78, or 79, or 80 amino acids in length. In one example, the sequence of the truncated human midkine protein can be 67 amino acids in length.

[0019] In one example, about 14, about 25, about 34, or about 58 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein (e.g., SEQ ID NO: 87). In one example, about 14 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. For example, 14 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises or consists of the sequence set forth in SEQ ID NO: 87. In another example, about 25 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. For example, 25 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises or consists of the sequence set forth in SEQ ID NO: 89. In a further example, about 34 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. For example, 34 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises or consists of the sequence set forth in SEQ ID NO:91. In another example, about 58 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. For example, 58 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises or consists of the sequence set forth in SEQ ID NO:95.

[0020] In one example, about 1 to 3 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 4 to 6 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 7 to 9 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 10 to 12 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 13 to 15 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 16 to 18 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 19 to 21 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein. In another example, about 22 to 24 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 25 to 27 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 30 to 32 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 33 to 35 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 36 to 38 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 48 to 51 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, about 52 to 54 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In another example, approximately 55 to 57 adjacent amino acids at the C-terminus of the human midkine protein are absent in the truncated human midkine protein.

[0021] In one example, the truncated human midkine protein comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 87, 89, 91, and 95.

[0022] For example, a truncated human midkine protein comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 87. In one example, the truncated human midkine protein comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 87. In another example, the truncated human midkine protein consists of an amino acid sequence having the sequence set forth in SEQ ID NO: 87.

[0023] For example, a truncated human midkine protein comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 89. In one example, the truncated human midkine protein comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 89. In another example, the truncated human midkine protein consists of an amino acid sequence having the sequence set forth in SEQ ID NO: 89.

[0024] For example, a truncated human midkine protein comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 91. In one example, the truncated human midkine protein comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 91. In another example, the truncated human midkine protein consists of an amino acid sequence having the sequence set forth in SEQ ID NO: 91.

[0025] For example, a truncated human midkine protein comprises an amino acid sequence having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 95. In one example, the truncated human midkine protein comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 95. In another example, the truncated human midkine protein consists of an amino acid sequence having the sequence set forth in SEQ ID NO: 95.

[0026] In one example, the composition comprises a polynucleotide encoding a truncated human midkine protein of the present disclosure described herein. According to this example, the sequence corresponding to exon 4 (e.g., as set forth in SEQ ID NO: 71 or 72) is absent or partially absent from the polynucleotide.

[0027] In one example, the polynucleotide is mRNA. According to this example, the sequence set forth in SEQ ID NO: 72 is absent or partially absent from the polynucleotide. For example, at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the sequence set forth in SEQ ID NO: 72 may be absent from the polynucleotide encoding the truncated midkine protein. In one example, a polynucleotide encoding a truncated human midkine protein of the present disclosure comprises an RNA sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 77. In one example, a polynucleotide encoding a truncated midkine protein comprises the RNA sequence set forth in SEQ ID NO:77.

[0028] In instances where one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary are present in the truncated midkine protein of the present disclosure, nucleotides encoding those additional amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 adjacent amino acids) may also be present in the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 81 may be present, or may be substantially present, in the mRNA sequence encoding the truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 81 is present in the mRNA sequence encoding the truncated midkine protein of the present disclosure.

[0029] Alternatively, or in addition, if one or more amino acids in exon 4 adjacent to the exon 4 / exon 5 boundary are present in a truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 82 may be present, or substantially present, in an mRNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 82 is present in an mRNA sequence encoding a truncated midkine protein of the present disclosure.

[0030] Alternatively, or in addition, if one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary are absent in the truncated midkine protein of the present disclosure, the nucleotides encoding those additional amino acids (e.g., nucleotides encoding 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids) may also be absent from the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure.

[0031] In one example, the polynucleotide is an mRNA and comprises the sequence of SEQ ID NO: 96, which has a deletion of about 42, 75, 102, or 174 contiguous nucleotides from the C-terminus of human midkine mRNA that are absent in the truncated human midkine mRNA. For example, the mRNA comprises the sequence set forth in SEQ ID NO: 96, which has a deletion of about 42 contiguous nucleotides from the C-terminus of human midkine mRNA that are absent in the truncated human midkine mRNA. In another example, the mRNA comprises the sequence set forth in SEQ ID NO: 96, which has a deletion of about 75 contiguous nucleotides from the C-terminus of human midkine mRNA that are absent in the truncated human midkine mRNA. In a further example, the mRNA comprises the sequence set forth in SEQ ID NO: 96, which has a deletion of about 102 contiguous nucleotides from the C-terminus of human midkine mRNA that are absent in the truncated human midkine mRNA. In another example, the mRNA comprises the sequence shown in SEQ ID NO: 96, which has a deletion of approximately 174 contiguous nucleotides at the C-terminus of human midkine mRNA that are absent in the truncated human midkine mRNA.

[0032] In one example, the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 14 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:86.

[0033] For example, the polynucleotide comprises an mRNA sequence having at least 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 86. In one example, the polynucleotide comprises an mRNA sequence having the sequence set forth in SEQ ID NO: 86. In such an example, the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 87.

[0034] In one example, the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 25 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:88.

[0035] For example, the polynucleotide comprises an mRNA sequence having at least 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 88. In one example, the polynucleotide comprises an mRNA sequence having the sequence set forth in SEQ ID NO: 88. In such an example, the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 89.

[0036] In one example, the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 34 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:90.

[0037] For example, the polynucleotide comprises an mRNA sequence having at least 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 90. In one example, the polynucleotide comprises an mRNA sequence having the sequence set forth in SEQ ID NO: 90. In such an example, the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 91.

[0038] In one example, the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 58 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:94.

[0039] For example, the polynucleotide comprises an mRNA sequence having at least 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 94. In one example, the polynucleotide comprises an mRNA sequence having the sequence set forth in SEQ ID NO: 94. In such an example, the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 95.

[0040] In another example, the polynucleotide is a DNA sequence. According to this example, the sequence set forth in SEQ ID NO: 71 is absent or partially absent from the polynucleotide. For example, at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the sequence set forth in SEQ ID NO: 71 may be absent from the polynucleotide encoding the truncated midkine protein. In one example, the polynucleotide comprises a DNA sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 76. In one example, the polynucleotide encoding the truncated midkine protein comprises the DNA sequence set forth in SEQ ID NO:76.

[0041] In instances where one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary are present in the truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 adjacent amino acids) may also be present in the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 79 may be present, or may be substantially present, in the DNA sequence encoding the truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 79 is present in the DNA sequence encoding the truncated midkine protein of the present disclosure.

[0042] Alternatively, or in addition, if one or more amino acids in exon 4 adjacent to the exon 4 / exon 5 boundary are present in a truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 80 may be present or substantially present in a DNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 80 is present in a DNA sequence encoding a truncated midkine protein of the present disclosure.

[0043] Alternatively, or in addition, if one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary are absent in the truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids) may also be absent from the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure.

[0044] In any example where the polynucleotide is a DNA sequence, the DNA sequence may be operably linked to a promoter and / or may be contained within an expression vector. For example, the polynucleotide is a DNA sequence encoding a truncated midkine protein, and the polynucleotide is operably linked to a promoter. In one example, the polynucleotide is a DNA sequence encoding a truncated midkine protein, and the polynucleotide is contained within an expression vector.

[0045] In any instance where the polynucleotide is a DNA sequence, the DNA sequence may be codon optimized.

[0046] The compositions of the present disclosure may further comprise one or more pharmaceutically acceptable carriers or diluents. In some examples, the compositions further comprise lipid nanoparticles (LNPs). In some examples, the polynucleotide is mRNA formulated with LNPs.

[0047] In an example where the composition contains an mRNA encoding a truncated midkine protein of the present disclosure, the mRNA can be encapsulated in LNPs. Alternatively, the mRNA encoding a truncated midkine protein of the present disclosure can be bound to LNPs. For example, the mRNA can be absorbed onto LNPs.

[0048] In an example where the composition comprises a truncated midkine protein of the present disclosure, the truncated protein can be encapsulated in LNPs. Alternatively, the truncated midkine protein can be bound to LNPs. For example, the truncated midkine protein can be absorbed onto LNPs.

[0049] In one example, the LNP further comprises a PEG-lipid, a structured lipid, and / or a neutral lipid. For example, the LNP further comprises a PEG-lipid. For example, the LNP further comprises a structured lipid. For example, the LNP further comprises a neutral lipid.

[0050] In one example, the LNP comprises an ionizable lipid. For example, the ionizable lipid is a cationic lipid. For example, the ionizable lipid is a zwitterionic lipid.

[0051] In one example, the LNP does not include an ionizable lipid.

[0052] The present disclosure also provides methods for producing a truncated human midkine protein or exosomes containing the truncated human midkine protein described herein, the method comprising culturing cells in the presence of one or more SSOs described herein for a time and under conditions sufficient for the cells to produce the truncated human midkine protein. In some examples, the method further comprises recovering the truncated human midkine protein or exosomes containing the truncated human midkine protein from the culture. In some examples, the method further comprises purifying the truncated human midkine protein or exosomes containing the truncated human midkine protein from the culture. In one example, the method comprises culturing cells in the presence of one or more SSOs selected from the group consisting of SEQ ID NOs: 60, 61, and 65.

[0053] The present disclosure also provides a method for producing a truncated human midkine protein or an exosome containing a truncated human midkine protein disclosed herein, the method comprising culturing cells containing a polynucleotide encoding a truncated human midkine protein disclosed herein for a time and under conditions sufficient for the cells to produce the truncated human midkine protein.

[0054] The present disclosure also provides a method for producing a truncated human midkine protein disclosed herein, comprising culturing cells containing a polynucleotide encoding a truncated human midkine protein disclosed herein for a time and under conditions sufficient for the cells to produce the truncated human midkine protein.

[0055] The present disclosure also provides a method for producing a truncated human midkine protein disclosed herein, comprising culturing a cell containing a composition disclosed herein for a time and under conditions sufficient for the cell to produce the truncated human midkine protein.

[0056] The present disclosure also provides a method for producing a truncated human midkine protein, comprising transfecting a cell with a polynucleotide disclosed herein under conditions sufficient for the cell to produce the truncated human midkine protein, and optionally recovering the truncated human midkine protein from the culture.

[0057] In some examples, the method further comprises recovering the truncated human midkine protein from the culture. In some examples, the method further comprises purifying the truncated human midkine protein or exosomes containing the truncated human midkine protein from the culture.

[0058] In some instances, inhibition of the interaction between human midkine and its ligand reduces cell migration.

[0059] In some examples, inhibition of the interaction between human midkine and its ligand reduces survival of cancer cells. For example, the cancer is a midkine-associated cancer. In one example, the cancer is breast cancer. In another example, the cancer is liver cancer.

[0060] The present disclosure also provides a method for inhibiting the interaction between human midkine and its ligand on the surface of or within a cell, the method comprising exposing the cell to a composition described herein.

[0061] The present disclosure also provides a method for inhibiting human midkine activity in a cell, the method comprising exposing the cell to a composition described herein.

[0062] The present disclosure also provides a method for treating or preventing a midkine-associated disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition described herein.

[0063] The present disclosure also provides use of the compositions described herein in the preparation of a medicament for treating or preventing a midkine-associated disease or disorder selected from an autoimmune disease, cancer, or inflammatory disease in a subject in need thereof. In some examples, the subject is being or will be treated with a chemotherapeutic agent or immunotherapy.

[0064] The present disclosure also provides uses of the compositions described herein for treating or preventing a midkine-associated disease or disorder selected from an autoimmune disease, cancer, or inflammatory disease in a subject in need thereof.

[0065] In some examples, the cancer is selected from the group consisting of esophageal cancer, thyroid cancer, bladder cancer, colorectal cancer, cutaneous and uveal melanoma, squamous cell carcinoma, osteosarcoma B-cell malignancies, leukemia, head and neck cancer, gallbladder cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, prostate cancer, and Wilms' tumor. For example, the cancer is liver cancer. For example, the cancer is breast cancer.

[0066] In one example, the subject to whom the composition of the present disclosure is administered is already undergoing treatment with another therapeutic agent for treating a midkine-related disease or disorder. For example, the subject and / or the midkine-related disease or disorder being treated may be refractory or resistant to treatment with other agents known to treat midkine-related diseases or disorders. In one example, the other agents known to treat midkine-related diseases or disorders are chemotherapeutic agents or immunotherapies.

[0067] In another example, the compositions of the present disclosure are administered in combination with another therapeutic agent known to treat a midkine-associated disease or disorder, ie, as an adjunct therapy.

[0068] Treatment of a midkine-associated disease or disorder according to any of the examples described herein may include one or more of inhibiting, reducing, or preventing midkine activity in a subject and / or reducing the severity of symptoms associated with a midkine-associated disease or disorder. In one example, a pharmaceutical agent will reduce midkine gene transcripts in a subject to which the pharmaceutical agent is administered.

[0069] Examples of midkine-associated diseases or disorders that can be inhibited, treated, or prevented include, but are not limited to, autoimmune diseases, cancer, or inflammatory diseases. In one example, the midkine-associated disease or disorder is cancer. In another example, the midkine-associated disease or disorder is an inflammatory disease. [Brief explanation of the drawings]

[0070] [Figure 1A] a) The nomenclature of the SSO is shown. The SSO name contains information about the gene, species, exon number, and sequence coordinates relative to the splice donor and acceptor sites. b) The nomenclature of the PCR primer is shown. The PCR primer ID contains the species, gene, target, primer direction, and additional information. [Figure 1B] (As mentioned above.) [Figure 2] Midkine (MDK) transcript reported on Ensembl. Each box represents an exon, and solid black lines represent introns. The sides of the chevrons indicate exons bounded by partial codons. Primers are shown in purple. [Figure 3] The midkine genomic sequence (NC_000011.10) is annotated with exon regions numbered based on the MANE-selected transcript T203 / transcript variant 3. Primers are shown in purple. [Figure 4] Optimization of six PCR primer sets by modifying PCR method, annealing temperature, number of cycles, and cell type. [Figure 5-1]Figure 1 shows the locations of exon splice enhancer (ESE) and exon splice silencer (ESS) motifs within midkine mRNA predicted by SpliceAid, as well as SSO annealing sites targeted to remove a) exon 3 and b) exon 4. Relative predicted splice factor binding site motif scores are shown on the y-axis, with positive values above the mRNA sequence indicating splice enhancer motifs, while negative values indicate splice silencer motifs. Exon sequences are shown in uppercase, and intron sequences are shown in lowercase. First-generation 2'OMe-PS SSOs are shown in black, second-generation microwalking SSOs are shown in blue, and single 20-mer SSOs are shown in purple. [Figure 5-2] (As mentioned above.) [Figure 6-1] Evaluation of SSO in Huh7 cells is provided. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with SSOs targeting a) exon 3 and b) exon 4 of midkine mRNA. Transfection concentrations (50–200 nM) are indicated above the gel images. Relative abundance (%) of amplicons is shown in graphs next to each gel image. GTC, Gene Tools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar; i, intron. [Figure 6-2] (As mentioned above.) [Figure 7-1] Evaluation of two SSO cocktails in Huh7 cells is provided. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with two SSO cocktails targeting a) exon 3 and b) exon 4 of midkine mRNA. Transfection concentrations are indicated above the gel images. Relative abundance (%) of amplicons is shown in the graph below each gel image. GTC, Gene Tools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar; i, intron. [Figure 7-2] (As mentioned above.) [Figure 8] Figure 1 provides an assessment of lower SSO concentrations in Huh7 cells. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with SSOs targeting a) exon 3 and b) exon 4 of midkine. Transfection concentrations are indicated above the gel images. The relative abundance (%) of amplicons lacking exon 3 (Δ3) or exon 4 (Δ4) is indicated below each gel image. GTC, Gene Tools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 9-1] Figure 1 provides an evaluation of SSOs microwalked around the original sequence in Huh7 cells. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with SSOs targeting a) exon 3 and b) exon 4 of midkine. The original sequence is shown in black, the microwalking sequence in blue, and the 20-mer sequence in purple. Transfection concentrations are indicated above the gel images. Relative abundance (%) of amplicons is shown in the graph below each gel image. GTC, Gene Tools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 9-2] (As mentioned above.) [Figure 10-1] Figure 1 provides an evaluation of the exon 3 + 4 SSO cocktail in Huh7 cells. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with a cocktail of one SSO targeting exon 3 and one targeting exon 4 of midkine. Transfection concentrations are indicated above the gel images. Relative abundance (%) of amplicons is shown in the graph below each gel image. GTC, Gene Tools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar; i, intron. [Figure 10-2] (As mentioned above.) [Figure 11-1] Evaluation of SSOs in SHSY5Y cells is provided. RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with individual SSOs targeting a) exon 3 and b) exon 4 of midkine, or c) a cocktail of two SSOs. Transfection concentrations are indicated above the gel images. Relative abundance (%) of amplicons is shown in graphs next to each gel image. GTC, Gene Tools control; L3K, Lipofectamine 3000 transfection reagent; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar; i, intron. [Figure 11-2] (As mentioned above.) [Figure 11-3] (As mentioned above.) [Figure 11-4] (As mentioned above.) [Figure 12] Evaluation of promising SSOs in HMC-1 cells is provided. a) RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with the most promising SSOs targeting exons 3 and 4 of midkine. Transfection concentrations are indicated above the gel images. b) The SMN positive control indicates transfection efficiency. Relative abundance (%) of amplicon is indicated below each gel image. GTC, Gene Tools control; L3K, Lipofectamine 3000 transfection reagent control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. c) Amplification of HMC-1 RNA treated with ezDNase or left untreated without a reverse transcription step to confirm effective removal of contaminating gDNA. [Figure 13]Further evaluation of promising SSOs in HMC-1 cells is provided. a) RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with the most promising SSOs targeting exons 3 and 4 of midkine. Transfection concentrations are indicated above the gel images. b) SMN positive controls indicate transfection efficiency. Relative abundance (%) of amplicons is indicated below each gel image. GTC, Gene Tools control; L3K, Lipofectamine 3000 transfection reagent; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 14] Evaluation of promising exon 4 microwalking SSOs in HMC-1 cells is provided. a) RT-PCR analysis of midkine transcripts after Lipofectamine 3000 transfection with the most promising SSOs microwalked around the original exon 4 sequence. Transfection concentrations are indicated above the gel images. b) SMN positive controls indicate transfection efficiency. Relative abundance (%) of amplicons is indicated below each gel image. GTC, GeneTools control; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 15] This figure provides an assessment of the effect of skipping at the pre-mRNA level in Huh7 cells transfected with the most promising 2'OMe-PS SSO targeting exons 3 and 4 of midkine using Lipofectamine 3000. The transfection concentration is indicated above the image. The relative abundance (%) of the amplicon is shown in the graph below the RT-PCR gel image. GTC, Gene Tools control; L3K, Lipofectamine 3000 transfection reagent; Neg, no template PCR control; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 16]This figure provides an evaluation of the effect of skipping with antisense oligonucleotides synthesized using phosphorodiamidate morpholino (PMO) nucleotide chemistry targeting the most promising regions of midkine exons 3 and 4. RT-PCR analysis of SHSY5Y cells transfected with one exon 3 and three exon 4 PMO SSOs using Neon transfection reagent is provided. Transfection concentrations are indicated above the images. Relative abundance (%) of amplicons is shown in the graph below the RT-PCR gel images. GTC, Gene Tools control; Zap, Neon transfection reagent; UT, untreated; bp, base pairs; FL, full-length amplicon; nM, nanomolar. [Figure 17] Further evaluation of SSO at the protein level is provided. Western blot analysis of Huh7 cells (a) and SHSY5Y cells (b) transfected with the most promising PMO AO targeting exons 3 and 4 of midkine using Lipofectamine 3000. Transfection concentrations are indicated above the images. GTC, Gene Tools control; L3K, Lipofectamine 3000 transfection reagent. [Figure 18] FIG. 1 is a representation of the full-length midkine protein sequence superimposed on the mRNA midkine sequence, including the 5' and 3' UTRs. The five exons are depicted in different colors, with the exact sequences with the exon boundaries indicated. [Figure 19] FIG. 1 is a graphic representation of a Western blot analysis of MDA-MB-231 breast cancer cells transfected with SEQ ID NOs: 86, 88, 90, 92, 94, and 96. Con UT = untransfected control; Con LMM = control Lipofectamine MessengerMax. [Figure 20] Graphical representation of Huh7 cell viability 72 hours after transfection with SEQ ID NOs: 86, 88, 90, 92, 94, and 96. LMM = control Lipofectamine MessengerMax; FL = full-length midkine. [Figure 21]Graphical representation of MDA-MD-231 cell viability 72 hours after transfection with SEQ ID NOs: 86, 88, 90, 92, 94, and 96. LMM = control Lipofectamine MessengerMax; FL = full-length midkine; GFP = green fluorescent protein. DETAILED DESCRIPTION OF THE INVENTION

[0071] Sequence table legend Sequence number 1 hMDK Ex1F SEQ ID NO: 2 hMDK Ex5R(o) SEQ ID NO: 3 hMDK Ex5R(i) SEQ ID NO: 4 hMDK Ex2F(o) SEQ ID NO: 5 hMDK Ex2F(i) SEQ ID NO: 6: Homo sapiens midkine full-length sequence SEQ ID NO: 7 Homo sapiens midkine domain 1 SEQ ID NO: 8 Homo sapiens midkine domain 2 SEQ ID NO: 9 Homo sapiens midkine domain 3 SEQ ID NO: 10 Homo sapiens midkine domain 4 SEQ ID NO: 11 Homo sapiens midkine domain 5 SEQ ID NO: 12 Homo sapiens midkine domain 6 SEQ ID NO: 13 Homo sapiens midkine domain 7 SEQ ID NO: 14 Homo sapiens midkine domain 8 SEQ ID NO: 15 Homo sapiens midkine domain 9 SEQ ID NO: 16 Homo sapiens midkine domain 10 SEQ ID NO: 17 Homo sapiens midkine domain 11 SEQ ID NO: 18 Homo sapiens midkine domain 12 SEQ ID NO: 19 Homo sapiens midkine domain 13 SEQ ID NO: 20 Homo sapiens midkine domain 14 SEQ ID NO: 21 Homo sapiens midkine domain 15 SEQ ID NO: 22 Homo sapiens midkine domain 16 SEQ ID NO: 23 Homo sapiens midkine domain 17 SEQ ID NO: 24 Homo sapiens midkine domain 18 SEQ ID NO: 25 Homo sapiens midkine domain 19 SEQ ID NO: 26 Homo sapiens midkine domain 20 SEQ ID NO: 27 Homo sapiens midkine domain 21 SEQ ID NO: 28 Homo sapiens midkine domain 22 SEQ ID NO: 29 Homo sapiens midkine domain 23 SEQ ID NO: 30 Homo sapiens midkine domain 24 SEQ ID NO: 31 Homo sapiens midkine domain 25 SEQ ID NO: 32 Homo sapiens midkine domain 26 SEQ ID NO: 33 Homo sapiens midkine domain 27 SEQ ID NO: 34 Homo sapiens midkine domain 28 SEQ ID NO: 35 Homo sapiens midkine domain 29 SEQ ID NO: 36 Homo sapiens midkine domain 30 SEQ ID NO: 37 Homo sapiens midkine domain 31 SEQ ID NO: 38 Homo sapiens midkine domain 32 SEQ ID NO: 39 Homo sapiens midkine domain 33 SEQ ID NO: 40 Homo sapiens midkine domain 34 SEQ ID NO: 41 Homo sapiens midkine domain 35 SEQ ID NO: 42 Homo sapiens midkine domain 36 SEQ ID NO: 43 MDK H3A(-12+13) SEQ ID NO: 44 MDK H3A(-04+21) SEQ ID NO: 45 MDK H3A(+89+113) SEQ ID NO: 46 MDK H3A(+143+167) SEQ ID NO: 47 MDK H3A(+146+165) SEQ ID NO: 48 MDK H3D(+15-10) SEQ ID NO: 49 MDK H4A(-12+13) SEQ ID NO: 50 MDK H4A(-04+21) SEQ ID NO: 51 MDK H4A(+74+98) SEQ ID NO: 52 MDK H4A(+106+130) SEQ ID NO: 53 MDK H4D(+10-15) SEQ ID NO: 54 MDK H3A(+133+157) SEQ ID NO: 55 MDK H3A(+138+162) SEQ ID NO: 56 MDK H3D(+21-04) SEQ ID NO: 57 MDK H3D(+10-15) SEQ ID NO: 58 MDK H3D(+05-20) SEQ ID NO: 59 MDK H4A(+69+93) SEQ ID NO: 60 MDK H4A(+79+103) SEQ ID NO: 61 MDK H4A(+101+125) SEQ ID NO: 62 MDK H4A(+111+135) SEQ ID NO: 63 MDK H4D(+15-10) SEQ ID NO: 64 MDK H4D(+05-20) SEQ ID NO: 65 MDK H4A(+100+124) SEQ ID NO: 66 RNA sequence for exon 1 of human midkine-5'UTR SEQ ID NO: 67 RNA sequence for exon 2 of human midkine SEQ ID NO: 68: Amino acid sequence corresponding to exon 2 of human midkine SEQ ID NO: 69 RNA sequence for exon 3 of human midkine SEQ ID NO: 70: Amino acid sequence corresponding to exon 3 of human midkine SEQ ID NO: 71 DNA sequence for exon 4 of human midkine SEQ ID NO: 72 RNA sequence for exon 4 of human midkine SEQ ID NO: 73: Amino acid sequence corresponding to exon 4 of human midkine SEQ ID NO: 74 RNA sequence for exon 5 of human midkine - C-terminus and 3'UTR SEQ ID NO: 75: Full-length coding region midkine protein sequence (excluding signal peptide) SEQ ID NO: 76: DNA sequence encoding a truncated midkine lacking exon 4 SEQ ID NO: 77: mRNA sequence for a truncated form of midkine lacking exon 4 SEQ ID NO: 78: Amino acid sequence for a truncated midkine in which the sequence corresponding to exon 4 is absent SEQ ID NO: 79: DNA sequence for the 5' fragment of exon 4 of human midkine SEQ ID NO: 80 DNA sequence for the 3' fragment of exon 4 of human midkine SEQ ID NO: 81 RNA sequence for the 5' fragment of exon 4 of human midkine SEQ ID NO: 82 RNA sequence for the 3' fragment of exon 4 of human midkine SEQ ID NO: 83: Amino acid sequence corresponding to the 5' fragment of exon 4 of human midkine SEQ ID NO: 84: Amino acid sequence corresponding to the 3' fragment of exon 4 of human midkine SEQ ID NO: 85: Amino acid sequence corresponding to exon 5 of human midkine SEQ ID NO: 86: RNA sequence corresponding to midkine Δ14 SEQ ID NO: 87: Amino acid sequence corresponding to midkine Δ14 SEQ ID NO: 88 RNA sequence corresponding to midkine Δ25 SEQ ID NO: 89: Amino acid sequence corresponding to midkine Δ25 SEQ ID NO: 90 RNA sequence corresponding to midkine Δ34 SEQ ID NO: 91: Amino acid sequence corresponding to midkine Δ34 SEQ ID NO: 92 RNA sequence corresponding to midkine Δ41 SEQ ID NO: 93: Amino acid sequence corresponding to midkine Δ41 SEQ ID NO: 94 RNA sequence corresponding to midkine Δ58 SEQ ID NO: 95: Amino acid sequence corresponding to midkine Δ58 SEQ ID NO: 96: RNA sequence corresponding to the coding region of full-length midkine, including the natural initiation codon and peptide leader sequence.

[0072] General Techniques and Definitions Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be taken to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0073] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more of such steps or features.

[0074] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0075] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the present disclosure, unless specifically stated otherwise.

[0076] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0077] Unless otherwise indicated, the recombinant protein, cell culture, molecular biology, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0078] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be taken as providing explicit support for both meanings or either meaning.

[0079] As used in this disclosure, including the claims, the words "a" and "an" refer to "one or more."

[0080] As used herein, the terms "about" and "approximately" are interchangeable and should be understood to generally refer to a range of numbers surrounding a given number, as well as all numbers within the range of recited numbers. Furthermore, all numerical ranges herein should be understood to include each whole integer within the range. Unless specified to the contrary, the terms "about" and "approximately" refer to + / - 10%, more preferably + / - 5%, and more preferably + / - 1% of the specified value. When used herein in the context of truncated human midkine protein sequences, "about" will be understood to refer to one amino acid on either side of the recited number.

[0081] As used herein, the terms "for example" and "i.e." are used without limitation and by way of example only, and should not be construed as referring only to those items explicitly listed in the present disclosure.

[0082] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the specified element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0083] Truncated midkine protein As described herein, the present disclosure provides truncated human midkine proteins in which the amino acid sequences encoded by exon 4 and / or exon 5 of human midkine are absent, partially absent, or substantially absent. The truncated forms of midkine proteins of the present disclosure are non-functional but retain the ability to form heterodimers with full-length functional midkine protein. In this manner, the truncated midkine proteins can form heterodimers with full-length functional midkine protein and inhibit the activity of full-length human midkine in cells or tissues and / or inhibit the interaction between human midkine and its ligand on the surface of or within cells.

[0084] References herein to "human midkine protein," "human MDK protein," or the like shall be understood to mean the native, full-length form of the human midkine protein. For purposes of nomenclature only, and without limitation, an exemplary sequence of the human midkine protein is represented in NCBI Reference Sequence: BC011704.2 (and represented in SEQ ID NO: 6).

[0085] The term "truncated" as used in the context of midkine protein refers to a midkine protein in which a portion of the protein is absent, resulting in a protein length that is shortened relative to the native, full-length human midkine protein. Thus, a truncated protein will be understood to contain less than the complete number of amino acids found in the native protein. While the present disclosure is not limited to any particular specific length of a truncated protein, it is contemplated that the amino acid sequence encoded by exon 4 of human midkine is absent, or partially or substantially absent, from the truncated form. In this regard, it is contemplated that the present disclosure may encompass truncated midkine proteins of any length in which the amino acid sequence encoded by exon 4 of human midkine is absent, partially absent, or substantially absent, provided that the truncated protein interferes with the activity of the full-length native midkine protein (e.g., by forming heterodimers therewith or otherwise inhibiting native midkine from binding to or interacting with a ligand).

[0086] As used herein, the term "native protein" refers to a protein inside or on the surface of a cell that is in its native or natural state, unmodified, properly folded, assembled, and / or secreted, and is operational and functional.

[0087] The term "substantially absent" as used in the context of the midkine protein sequences of the present disclosure shall be understood to mean that almost all of the referenced sequence is absent. For example, a truncated midkine protein substantially absent from the amino acid sequence encoded by exon 4 of human midkine contains less than 30% of the amino acid sequence encoded by exon 4 of human midkine. For example, a truncated midkine protein substantially absent from the amino acid sequence encoded by exon 4 of human midkine contains less than 20% of the amino acid sequence encoded by exon 4 of human midkine. Preferably, a truncated midkine protein substantially absent from the amino acid sequence encoded by exon 4 of human midkine contains less than about 10% of the amino acid sequence encoded by exon 4 of human midkine. For example, a truncated midkine protein substantially absent from the amino acid sequence encoded by exon 4 of human midkine may contain fewer than 10 (e.g., fewer than 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1) amino acids encoded by exon 4 of human midkine. Preferably, a truncated midkine protein substantially absent from the amino acid sequence encoded by exon 4 of human midkine is non-functional.

[0088] The term "partially absent" as used in the context of the midkine protein sequences of the present disclosure shall be understood to mean that a portion of the referenced sequence is absent. For example, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent contains about 60% of the amino acid sequence encoded by exon 4 of human midkine. For example, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent contains about 70% of the amino acid sequence encoded by exon 4 of human midkine. In another example, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent contains about 80% of the amino acid sequence encoded by exon 4 of human midkine. In another example, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent contains about 90% of the amino acid sequence encoded by exon 4 of human midkine. For example, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent may contain fewer than 10 (e.g., 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or less than 1) amino acids encoded by exon 4 of human midkine. Preferably, a truncated midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is partially absent is non-functional.

[0089] The term "substantially present" as used in the context of the midkine protein sequences of the present disclosure shall be understood to mean that almost all of the referenced sequence is present.

[0090] As described herein, the amino acid sequence encoded by exon 4 of human midkine may be completely absent from the truncated midkine proteins of the present disclosure. In some examples, the amino acid sequence encoded by exon 5 of human midkine may be absent from the truncated midkine proteins of the present disclosure. In such examples, the truncated midkine proteins of the present disclosure may be designed so that they do not have C-terminal amino acids. According to such examples, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, or about 59 contiguous amino acids from the C-terminus of the human midkine protein are absent in the truncated human midkine protein. In one example, about 14 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises the amino acid sequence set forth in SEQ ID NO: 87. In another example, about 25 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises the amino acid sequence set forth in SEQ ID NO: 89. In another example, about 34 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises the amino acid sequence set forth in SEQ ID NO: 91. In one example, about 58 contiguous amino acids at the C-terminus of the human midkine protein are absent from the truncated human midkine protein. In such an example, the truncated human midkine protein comprises the amino acid sequence set forth in SEQ ID NO: 95.

[0091] The amino acid sequence encoded by exon 4 of human midkine is set forth in SEQ ID NO: 73. According to certain examples in which the amino acid sequence encoded by exon 4 of the human midkine protein is substantially absent, at least about 70% (e.g., at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) of the sequence set forth in SEQ ID NO: 73 is absent from the truncated human midkine protein. In another example, the amino acid sequence encoded by exon 4 of the human midkine protein (i.e., the amino acid sequence set forth in SEQ ID NO: 73) is absent from the truncated human midkine protein.

[0092] In examples where the amino acid sequence encoded by exon 4 is substantially absent, one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary may also be present in the truncated midkine protein. For example, 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 83 may be present in the truncated human midkine protein. In some examples, the amino acid sequence set forth in SEQ ID NO: 83 is present in the truncated human midkine protein. Alternatively, or in addition, when the amino acid sequence encoded by exon 4 is substantially absent, one or more amino acids within exon 4 adjacent to the exon 4 / exon 5 boundary may be present. For example, 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 84 may be present in the truncated human midkine protein. In yet another example, one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary may also be present in a truncated midkine protein, and one or more amino acids within exon 4 adjacent to the exon 4 / exon 5 boundary may be present in a truncated human midkine protein. For example, 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 83 may be present in a truncated human midkine protein, and 1, 2, 3, 4, or 5 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 84 may be present in a truncated human midkine protein. In one example, the amino acid sequences set forth in SEQ ID NOs: 83 and 84 may be present in a truncated human midkine protein.

[0093] Alternatively, or in addition, one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary may be absent from the truncated midkine protein. For example, 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids of the amino acid sequence set forth in SEQ ID NO: 85 may be absent, partially absent, or substantially absent from the truncated human midkine protein. In some examples, the amino acid sequence set forth in SEQ ID NO: 85 is absent from the truncated human midkine protein.

[0094] In one example, the truncated human midkine protein comprises an amino acid sequence having at least about 70% (e.g., at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity to the sequence set forth in SEQ ID NO:78. In another example, the truncated human midkine protein comprises the sequence set forth in SEQ ID NO:78. In one example, the truncated human midkine protein consists of the sequence set forth in SEQ ID NO:78.

[0095] In some examples, truncated human midkine proteins in which the amino acid sequence corresponding to exon 4 is absent, partially absent, or substantially absent are about 60 to about 80 amino acids in length. For example, the sequence of the truncated human midkine protein can be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids in length. In one example, the sequence of the truncated human midkine protein can be 67 amino acids in length.

[0096] Polypeptide production The truncated midkine proteins of the present disclosure can be produced by any means known in the art for producing polypeptides.

[0097] In one example, the truncated midkine proteins of the present disclosure are synthesized using any chemical method known to those skilled in the art. For example, synthetic proteins can be prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and / or unnatural amino acids.

[0098] In another example, the truncated midkine proteins of the present disclosure can be expressed by recombinant means, for example, a nucleic acid encoding a truncated midkine protein can be placed in operable linkage with a promoter or other regulatory sequence capable of controlling expression in a cell system or organism.

[0099] Typical promoters suitable for expression in bacterial cells include, for example, the lacz promoter, Ipp promoter, temperature-sensitive λL or λR promoter, T7 promoter, T3 promoter, SP6 promoter, or semi-artificial promoters, such as the IPTG-inducible tac promoter or lacUV5 promoter. Several other gene construction systems for expressing nucleic acids encoding the truncated midkine proteins of the present disclosure in bacterial cells are well known in the art and are described, for example, in Ausubel et al. (1988) and Sambrook et al. (2001).

[0100] A number of expression vectors have been described for the expression of recombinant polypeptides in bacterial cells, including, inter alia, PKC3, pKK173-3, pET28, pCR Vector Suite (Invitrogen), pGEM-T Easy Vector (Promega), pL Expression Vector Suite (Invitrogen), or the pBAD / Thio-TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen).

[0101] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including, for example, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0102] Examples of expression vectors for expression in yeast cells include pACT vectors (Clontech), pDBleu-X vectors, pPIC Vector Suite (Invitrogen), pGAPZ Vector Suite (Invitrogen), pHYB vectors (Invitrogen), pYD1 vectors (Invitrogen), and pNMT1, pNMT41, pNMT81 TOPO vectors (Invitrogen), pPC86-Y vectors (Invitrogen), pRH series vectors (Invitrogen), and pYESTrp series vectors (Invitrogen).

[0103] Expression vectors for expression in mammalian cells include, for example, the pcDNA Vector Suite (Invitrogen), the pTARGET series of vectors (Promega), and the pSV Vector Suite (Promega). However, many other expression vectors for mammalian cells are known in the art and are contemplated herein.

[0104] Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. 2001 and other laboratory textbooks. In one example, a nucleic acid encoding a truncated midkine protein of the present disclosure can be introduced into prokaryotic cells using, for example, electroporation or calcium chloride-mediated transformation. In another example, a nucleic acid can be introduced into mammalian cells using, for example, microinjection, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, e.g., by using lipofectamine (Invitrogen) and / or cellfectin (Invitrogen), PEG-mediated DNA uptake, electroporation, transduction with adenovirus, herpesvirus, togavirus, or retrovirus, and microparticle bombardment, e.g., by using DNA-coated tungsten or gold particles. Alternatively, a nucleic acid can be introduced into yeast cells using conventional techniques, such as, for example, electroporation and PEG-mediated transformation.

[0105] In yet another example, a truncated midkine protein of the present disclosure can be expressed by a cell or organism into which a splice-switching oligonucleotide of the present disclosure has been introduced, such that a truncated form of midkine is produced by the cell or organism. The truncated midkine protein can then be recovered.

[0106] Thus, the present disclosure also contemplates a method for producing truncated midkine proteins by the use of splice-switching oligonucleotides (SSOs) that target the pre-mRNA sequence of human midkine.

[0107] For example, the present disclosure provides a composition comprising a splice-switching oligonucleotide (SSO) that targets the pre-mRNA sequence of human midkine.

[0108] In some examples, the SSO described herein can be used to produce the truncated human midkine proteins described herein. In one example, the SSO can be used to produce the truncated human midkine proteins described herein in vitro. For example, the present disclosure includes providing the SSO described herein to an in vitro cell to produce a truncated human midkine protein, and optionally isolating the truncated human midkine protein from the cell.

[0109] In another example, the SSO can be used to produce in vivo the truncated human midkine proteins described herein. For example, the present disclosure includes providing the SSO to an in vivo cell or subject to produce in vivo the truncated human midkine proteins described herein.

[0110] The SSO of the present disclosure is 10 to 50 nucleotides in length (e.g., 20 to 25 nucleotides in length) and comprises a polynucleotide sequence of at least 10 contiguous nucleotides that is substantially complementary to a target region of corresponding length within the pre-mRNA sequence of human midkine.

[0111] As used herein, the terms "splice-switching oligonucleotide," "splice-switching oligomer," "SSO," and "antisense oligonucleotide," or "AO," when used in the context of splice switching, refer to short oligonucleotides that are substantially complementary to and base-pair with a fragment of a pre-mRNA sequence, thereby disrupting the normal splicing repertoire of a transcript by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. In doing so, the splice-switching oligonucleotide can induce targeted exon skipping. The use of SSOs for targeted exon skipping is well known in the art [see, e.g., G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)]. To distinguish between different antisense molecules, the AO nomenclature system has been proposed and published (Aung-Htut MT et al. 2019 Int J Mol Sci 20:5030) (see Figure 1).

[0112] The term "oligonucleotide" as used in the context of SSO refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), where the polymer or oligomer of monomers contains any combination of s or nucleosides, modified s or nucleosides, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). Oligonucleotides can be single-stranded or double-stranded, or a combination thereof. Single-stranded oligonucleotides can have a double-stranded region, and double-stranded oligonucleotides can have a single-stranded region (such as a microRNA or shRNA).

[0113] As used herein, "RNA" refers to a sequence comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., messenger RNA, and modified RNA that differs from naturally occurring RNA by one or more additions, deletions, substitutions, and / or modifications within the RNA sequence of the disclosure, and can also include non-naturally occurring or chemically synthesized or non-standard, such as deoxynucleotides.

[0114] An SSO and DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each sequence are occupied by polynucleotides that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an oligonucleotide and a DNA or RNA target. It is understood in the art that the sequence of an SSO need not be 100% complementary to the sequence of its target region to be specifically hybridizable. An SSO is specifically hybridizable if binding of the compound to the target DNA or RNA sequence interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the SSO to non-target regions under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0115] As used herein, the term "complementary" with respect to a sequence refers to the complement of the sequence by Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C) and adenine (A) pairs with either uracil (U) or thymine (T). A sequence can be complementary to the entire length of another sequence, or to a particular fragment or length of another sequence. Those of skill in the art will recognize that U can occur in RNA and T can occur in DNA. Thus, A in either an RNA or DNA sequence can pair with U in an RNA sequence or T in a DNA sequence.

[0116] As used herein, the term "substantially complementary" is used to indicate a sufficient degree of complementarity or exact match such that stable and specific binding occurs, for example, between an SSO and an SSO-complementary sequence, or between an SSO and a target region. That is, an SSO can "specifically hybridize" to its cognate target region. It is understood that a nucleic acid sequence need not be 100% complementary to its target or complement sequence. The term encompasses sequences that are complementary to another sequence, excluding overhangs. In some cases, a sequence is complementary to another sequence except for one to four mismatches. In some cases, a sequence is complementary except for one mismatch. In some cases, a sequence is complementary except for two mismatches. In other cases, a sequence is complementary except for three mismatches. In still other cases, a sequence is complementary except for four mismatches.

[0117] The SSO may be capable of hybridizing to its target region under physiological conditions, ie, under normal conditions within a cell.

[0118] As used herein, "target" or "target region" refers to a stretch of polynucleotide within the pre-mRNA sequence of human midkine to which an SSO of the present disclosure is substantially complementary (or complementary) and capable of hybridizing (e.g., under physiological conditions).

[0119] The length of the SSO of the present disclosure can be varied as long as the SSO can selectively bind to the intended position in the pre-mRNA sequence. In this regard, it is well known by those skilled in the art that it is possible to increase or decrease the length of the SSO and / or introduce mismatch bases without eliminating activity. Methods for determining the desired activity are disclosed herein and are well known to those skilled in the art.

[0120] Generally, an SSO will comprise a polynucleotide sequence from about 10 polynucleotides in length up to about 50 polynucleotides in length. For example, an SSO can be about 10 polynucleotides in length, or about 15 polynucleotides in length, or about 20 polynucleotides in length, or about 25 polynucleotides in length, or about 30 polynucleotides in length, or about 35 polynucleotides in length, or about 40 polynucleotides in length, or about 45 polynucleotides in length, or about 50 polynucleotides in length. In particular examples, the length of the SSO is 15-30 polynucleotides in length, e.g., 15-25 polynucleotides in length.

[0121] The SSOs of the present disclosure can be designed to disrupt splicing, thereby inducing exon skipping of exons 1, 2, 3, 4, or 5 of human midkine.

[0122] As used herein, "exon skipping" refers to modifying the processing of a pre-mRNA transcript so that the spliced mRNA sequence contains a different combination of exons as a result of exon skipping. In the context of the present disclosure, exon skipping refers to modifying the splicing of midkine pre-mRNA to achieve the deletion of one or more exons.

[0123] In one example, the SSO of the present disclosure is designed to specifically hybridize to a target region of corresponding length within the pre-mRNA sequence of human midkine to disrupt splicing, thereby inducing exon skipping of exon 3. The degree of identity of the SSO polynucleotide sequence to the target region should be at least 85%, 90%, 95%, or 100%. The SSO may, of course, contain unrelated sequences that may function to stabilize the sequences described herein.

[0124] According to this example, the SSO can comprise a polynucleotide sequence of at least 10 contiguous polynucleotides (e.g., 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25 contiguous polynucleotides), which is complementary to a region of corresponding length within the sequence set forth in any one of SEQ ID NOs: 7-14, 20-25, and 31-35, optionally except for 1, 2, 3, or 4 mismatches.

[0125] In one example, an SSO of the present disclosure is designed to specifically hybridize to a target region of corresponding length within the pre-mRNA sequence of human midkine to disrupt splicing, thereby inducing exon skipping of exon 4. According to this example, the SSO may comprise a polynucleotide sequence of at least 10 contiguous polynucleotides (e.g., 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25 contiguous polynucleotides), which is substantially complementary to a region of corresponding length within the sequence set forth in any one of SEQ ID NOs: 15-19, 26-30, and 36-42. For example, an SSO can comprise a polynucleotide sequence of at least 10 contiguous polynucleotides (e.g., 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25 contiguous polynucleotides), which is complementary to a region of corresponding length within the sequence set forth in any one of SEQ ID NOs: 15-19, 26-30, and 36-42, optionally except for 1, 2, 3, or 4 mismatches.

[0126] Typically, the SSO of the present disclosure will be synthesized in vitro. Methods for synthesizing oligonucleotides are known in the art. However, in some cases where modified polynucleotides and backbones are not required, the SSO of the present disclosure can be expressed in vitro or in vivo in a suitable system, such as a recombinant virus or cell.

[0127] After production / expression / synthesis, the truncated midkine proteins of the present disclosure can be recovered and / or purified using methods known in the art. For example, affinity purification may be used to purify any of the proteins of the present disclosure. Methods for isolating proteins using affinity chromatography are known in the art and are described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).

[0128] The degree of purity of any protein of the present disclosure may be determined by a variety of methods, including identification of a major large peak in HPLC or UPLC.

[0129] Polynucleotides The present disclosure also provides polynucleotides encoding the truncated human midkine proteins described herein.

[0130] As used herein, the term "polynucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), where a polymer or oligomer of polynucleotide monomers contains any combination of polynucleotides or nucleosides, modified polynucleotides or nucleosides, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). The term "polynucleotide" is used interchangeably herein with the terms "nucleic acid" and "oligonucleotide." The term "recombinant polynucleotide" refers to a polynucleotide comprising a nucleic acid sequence produced or arrived at by recombinant means.

[0131] As used herein, the terms "encode," "encodes," or "encoding" refer to a region of RNA (e.g., mRNA) that can be translated into a polypeptide or protein, or, when used in the context of DNA, a region of DNA that can be transcribed to produce an mRNA that can be translated into a polypeptide or protein.

[0132] As described herein, the present disclosure relates to polynucleotides encoding truncated human midkine proteins of the present disclosure (e.g., sequences corresponding to exon 4 and / or exon 5 are absent, partially absent, or substantially absent from the polynucleotide). The DNA sequence corresponding to exon 4 of the human midkine protein is set forth in SEQ ID NO: 71. The RNA sequence corresponding to exon 4 of the human midkine protein is set forth in SEQ ID NO: 72.

[0133] In one example, the polynucleotide is mRNA. For example, the mRNA can be conventional mRNA (cRNA) or self-amplifying RNA (sa-mRNA). In accordance with the example in which the polynucleotide is mRNA, the sequence set forth in SEQ ID NO: 72 is absent, partially absent, or substantially absent from the polynucleotide. For example, at least about 5% (e.g., at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%) of the sequence set forth in SEQ ID NO: 72 can be absent from the polynucleotide encoding the truncated midkine protein.

[0134] A polynucleotide encoding a truncated human midkine protein may comprise an mRNA sequence having at least about 70% (e.g., at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity to the sequence set forth in SEQ ID NO: 77. In one example, the polynucleotide encoding a truncated midkine protein comprises the RNA sequence set forth in SEQ ID NO: 77.

[0135] In examples where one or more amino acids encoded by exon 4 (adjacent to the exon 3 / exon 4 boundary) are present in a truncated midkine protein, i.e., where exon 4 is partially absent or substantially absent from the coding sequence, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 81 may be present, or may substantially be present, in an mRNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 81 is present in an mRNA sequence encoding a truncated midkine protein of the present disclosure.

[0136] Alternatively, or in addition, if one or more amino acids in exon 4 adjacent to the exon 4 / exon 5 boundary are present in a truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 82 may be present, or substantially present, in an mRNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 82 is present in an mRNA sequence encoding a truncated midkine protein of the present disclosure.

[0137] Alternatively, or in addition, if one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary are absent in the truncated midkine protein of the present disclosure, the nucleotides encoding those additional amino acids (e.g., nucleotides encoding 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids) may also be absent from the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure. In some examples, exon 5 of human midkine is absent from the polynucleotide encoding the truncated midkine protein.

[0138] According to one such example, the polynucleotide encodes a truncated human midkine protein comprising the amino acid sequence set forth in SEQ ID NO: 87. In another example, the polynucleotide encodes a truncated human midkine protein comprising the amino acid sequence set forth in SEQ ID NO: 89. In one example, the truncated human midkine protein comprises the amino acid sequence set forth in SEQ ID NO: 91. In one example, the polynucleotide encodes a truncated human midkine protein comprising the amino acid sequence set forth in SEQ ID NO: 95.

[0139] In other examples, the polynucleotide is a DNA sequence. In examples where the polynucleotide is a DNA sequence, the sequence set forth in SEQ ID NO: 71 is absent, partially absent, or substantially absent from the polynucleotide. For example, at least about 70% (e.g., at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) of the sequence set forth in SEQ ID NO: 71 may be absent from the polynucleotide encoding the truncated midkine protein. In one example, the polynucleotide comprises a DNA sequence having at least about 70% (e.g., at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity to the sequence set forth in SEQ ID NO: 76. In one example, the polynucleotide encoding the truncated midkine protein comprises the DNA sequence set forth in SEQ ID NO: 76.

[0140] In examples where one or more amino acids within exon 4 and adjacent to the exon 3 / exon 4 boundary are present in a truncated midkine protein of the present disclosure, i.e., where exon 4 is partially absent or substantially absent from the coding sequence, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 79 may be present, or may substantially be present, in a DNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 79 is present in a DNA sequence encoding a truncated midkine protein of the present disclosure.

[0141] Alternatively, or in addition, if one or more amino acids in exon 4 adjacent to the exon 4 / exon 5 boundary are present in a truncated midkine protein of the present disclosure, the nucleotides encoding those amino acids (e.g., nucleotides encoding 1, 2, 3, 4, or 5 contiguous amino acids) may also be present in a polynucleotide sequence encoding a truncated human midkine protein of the present disclosure. For example, the polynucleotide sequence set forth in SEQ ID NO: 80 may be present or substantially present in a DNA sequence encoding a truncated midkine protein of the present disclosure. In one example, the sequence set forth in SEQ ID NO: 80 is present in a DNA sequence encoding a truncated midkine protein of the present disclosure.

[0142] Alternatively, or in addition, if one or more of the amino acids within exon 5 and adjacent to the exon 4 / exon 5 boundary are absent in the truncated midkine protein of the present disclosure, the nucleotides encoding those additional amino acids (e.g., nucleotides encoding 1, 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids) may also be absent from the polynucleotide sequence encoding the truncated human midkine protein of the present disclosure.

[0143] In any example where the polynucleotide is a DNA sequence, the DNA sequence may be operably linked to a promoter and / or may be contained within an expression vector.

[0144] The DNA sequence may be a heterologous DNA sequence. The DNA sequence may comprise at least one DNA sequence or one or more heterologous DNA sequences.

[0145] The DNA sequence can be an optimized DNA sequence. Such optimization can increase the expression of the truncated midkine protein, particularly its biological effect (including neutralizing effect). Optimization can also improve transcription and / or translation. Optimization can include one or more of the following: a low GC content leader sequence to increase transcription, reduction of mRNA secondary structure and codon optimization, optimized 5' UTR and 3' UTR, optimized transcription termination signal, optimal Kozak sequence (e.g., GCC ACC) to increase translation efficiency, and elimination of cis-acting sequence motifs (e.g., internal TATA boxes) whenever possible.

[0146] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a polynucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0147] As used herein, the term "promoter" refers to a DNA element or sequence that can control transcription of a polynucleotide of the present disclosure into mRNA when the promoter is positioned at the 5' end of (i.e., preceding) the polynucleotide sequence. Thus, a promoter is typically located 5' (i.e., upstream) of the polynucleotide sequence whose transcription it controls into mRNA and provides a site for specific binding and initiation of transcription by RNA polymerase.

[0148] As used herein, the term "operably linked" or "operably linked" (or the like) means that a coding nucleic acid sequence (i.e., a polynucleotide encoding a truncated midkine protein) is linked to or in relation to a control sequence, e.g., a promoter, in a manner that facilitates expression of the coding sequence.

[0149] In addition to promoters, it is also contemplated that the polynucleotides of the present disclosure can be operably linked to other regulatory sequences, such as enhancers and other expression control elements, which are recognized in the art and can be selected to direct expression of the polynucleotide.

[0150] Suitable expression vectors and promoters that can be used with the polynucleotides of the present disclosure are described herein in the context of producing truncated midkine proteins and, unless specifically stated otherwise, shall be taken to apply mutatis mutandis to each and every example of the present disclosure describing polynucleotides.

[0151] However, in some instances, promoters useful in the present disclosure can be tissue- or cell-specific. The term "tissue-specific" as applied to a promoter refers to a promoter that can direct the selective transcription of a nucleic acid of interest to a particular type of tissue (e.g., liver or muscle) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., kidney). The term "cell-specific" as applied to a promoter refers to a promoter that can direct the selective transcription of a nucleic acid of interest in a particular type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue.

[0152] The polynucleotides of the present disclosure further comprise one or more strategically positioned multiple cloning sites and / or unique restriction sites so that the promoter, the sequence encoding the truncated midkine protein, and / or other regulatory element can be easily removed or replaced. Constructs containing these elements can be assembled from smaller oligonucleotide building blocks using strategically positioned restriction sites and / or complementary cohesive ends.

[0153] Polynucleotide production Suitable methods for the production of the polynucleotides of the disclosure will be apparent to those of skill in the art and / or are described herein.

[0154] For example, the generation of DNA sequences and / or constructs comprising same can be achieved using any suitable genetic engineering technique known in the art, including, but not limited to, standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing. When a construct comprising a polynucleotide of the present disclosure is a viral construct, the construct will include, for example, sequences necessary for packaging the polynucleotide of the present disclosure into viral particles and / or sequences that enable integration of the polynucleotide of the present disclosure into the genome of a target cell. In some examples, each viral construct will additionally contain genes that enable viral replication and propagation, with such genes being supplied in trans. Additionally, each viral construct can contain genes or gene sequences from the genome of any known organism, either natively integrated or modified. For example, the viral construct may include sequences useful for replication of the construct in bacteria.

[0155] The construct may also contain additional genetic elements. The types of elements that can be included in the construct are not limited in any way and can be selected by those skilled in the art. For example, the additional genetic elements may include a reporter gene, such as one or more genes for fluorescent marker proteins such as GFP or RFP, an easily assayed enzyme such as beta-galactosidase, luciferase, beta-glucuronidase, chloramphenicol acetyltransferase, or secreted embryonic alkaline phosphatase, or a protein for which an immunoassay is readily available, such as a hormone or cytokine.

[0156] Other genetic elements that may find use in embodiments of the present disclosure related to genetic constructs containing polynucleotides of the present disclosure include those encoding proteins that confer a selective growth advantage to cells, such as adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin B-phosphotransferase, and drug resistance, or those encoding proteins that provide biosynthetic capabilities missing from auxotrophs. When a reporter gene is included with the construct, it may contain an internal ribosome entry site (IRES) sequence. In one example, the additional genetic element is operably linked to and controlled by an independent promoter / enhancer. In addition, a suitable origin of replication for propagation of the construct in bacteria may be used. The origin of replication sequence is generally separate from the cargo polynucleotide sequence and other genetic sequences. Such origins of replication are known in the art and include the pUC, ColEl, 2-micron, or SV40 origins of replication.

[0157] In another example, the mRNA of the present disclosure can be produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. In one exemplary method, competent bacterial cells (e.g., Escherichia coli) are transformed with a DNA plasmid encoding the mRNA of the present disclosure. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in E. coli cultures.

[0158] Plasmid DNA is then isolated after fermentation. For example, the plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other routine methods known to those skilled in the art. After isolation, the plasmid DNA is linearized by restriction digestion (i.e., using a restriction enzyme). The restriction enzyme is removed using methods known in the art, including, for example, phenol / chloroform extraction and ethanol precipitation.

[0159] In another example, the mRNA of the present disclosure can be produced by in vitro transcription from a linearized DNA template using an RNA polymerase (e.g., T7 RNA polymerase). Following in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will understand that synthetic RNA capping is performed to modify mRNA processing and contribute to mRNA stabilization. In one example, the RNA is enzymatically 5'-capped. For example, the 5' cap is a Cap 0 structure or a Cap 1 structure. In one example, the 5' cap is a Cap 0 structure, e.g., a 5' cap (i.e., Cap) consisting of an inverted 7-methylguanosine connected to the rest of the RNA via a 5'-5' triphosphate bridge. In one example, the 5' cap is a Cap 1 structure, e.g., a 5' cap (i.e., Cap 1) consisting of a Cap 0 with an additional methylation at the 2'0 position of the initiating polynucleotide. Those skilled in the art will also understand that polyadenylation of mRNA can occur within mRNAs that contain polyadenylation sequences.

[0160] mRNA can also be purified. Various methods for purifying mRNA will be apparent to those skilled in the art. For example, mRNA is purified using lithium chloride (LiCl) precipitation. In another example, mRNA is purified using tangential flow filtration (TFF). In one example, mRNA is purified using anion exchange chromatography. For example, anion exchange chromatography is performed using an anion exchange resin (e.g., MustangQ® membrane (Pall®)). After purification, mRNA is resuspended, for example, in nuclease-free water.

[0161] Examination of candidate SSOs The SSOs of the present disclosure can be conveniently and routinely produced through well-known solid-phase synthesis techniques. Any other means for such synthesis known in the art can additionally or alternatively be used. It is well known to use similar techniques to prepare SSOs such as phosphorothioates and alkylated derivatives. In this regard, the present disclosure is not limited by the method of SSO synthesis.

[0162] Methods for SSO purification and analysis will also be known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray-mass spectrometry. Such synthesis and analysis methods can be performed in multi-well plates. The methods of the present disclosure are not limited by the SSO purification method.

[0163] Once synthesized, candidate SSOs can be tested for their desired activity using standard procedures and techniques known in the art. For example, screening of candidates can involve administering the candidate SSO to cells that express midkine in vitro (e.g., via transfection) and determining the effect of the candidate SSO on midkine expression in those cells at the mRNA and / or protein level. Detection and quantification of full-length and truncated forms of midkine protein can then be performed using standard molecular techniques such as protein gel electrophoresis and Western blotting. Similarly, detection and quantification of full-length and truncated forms of midkine mRNA can then be performed using mRNA reverse transcription PCR (RT-PCR) followed by agarose gel electrophoresis, as known in the art. In another example, candidate SSOs can be administered to animals (e.g., animal models of midkine-associated diseases or conditions known to express midkine), and the animals can be screened for the amount and species of expressed midkine mRNA and / or midkine protein. Functional assays may be performed to assess the effect of candidate SSOs on midkine function in animals to which they are administered. In another example, candidate SSOs may simply be tested for their ability to hybridize to a target polynucleotide (such as mRNA).

[0164] composition The present disclosure also provides compositions comprising the truncated human midkine proteins, polynucleotides encoding the truncated human midkine proteins, and / or SSOs described herein for administration. For example, the compositions may comprise one or more truncated human midkine proteins described herein. For example, the compositions may comprise one or more polynucleotides encoding the truncated human midkine proteins described herein. For example, the compositions may comprise one or more SSOs targeting human midkine described herein.

[0165] The compositions of the present disclosure may be mixed, encapsulated, conjugated (e.g., fused), or otherwise associated with other molecules, molecular structures, or mixtures of compounds to aid in uptake, distribution, and / or absorption, resulting in, for example, exosomes, liposomes, lipid nanoparticles (LNPs), receptor-targeting molecules, oral, rectal, topical, inhalable, injectable, or other formulations (Paunovska K et al (2022) Nature Reviews Genetics 4:1-16). Representative U.S. patents that teach the preparation of such uptake, distribution, and / or absorption-enhancing formulations include U.S. Pat. Nos. 5,108,921, 5,354,844, 5,416,016, 5,459,127, 5,521,291, 5,543,158, 5,547,932, 5,583,020, 5,591,721, 5,426,330, 5,534,899, 5,013,556, 5,108,921, 5,2 ... 13,804, US 5,227,170, US 5,264,221, US 5,356,633, US 5,395,619, US 5,416,016, US 5,417,978, US 5,462,854, US 5,469,854, US 5,512,295, US 5,527,528, US 5,534,259, US 5,543,152, US 5,556,948, US 5,580,575, and US 5,595,756.

[0166] As used herein, the term "lipid nanoparticle" or "LNP" is understood to refer to a lipid-based particle having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm). LNPs may contain ionizable cationic compounds, neutral lipids, charged lipids, sterols, and PEGylated lipids. In some examples, lipid nanoparticles or LNPs may be selected from liposomes or vesicles, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single, unilamellar, or 10-fold, multilamellar), micellar lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles. In some examples, lipid nanoparticles or LNPs may have a structure comprising a single monolayer or bilayer of lipids encapsulating a solid phase. In other examples, lipid nanoparticles or LNPs do not have an aqueous or other liquid phase within their interior.

[0167] The LNPs may comprise a bilayer stabilizing component (BSC) such as an ATTA lipid or a PEG lipid, e.g., PEG coupled to dialkyloxypropyl (PEG-DAA) as described in WO 05 / 026372, PEG coupled to diacylglycerol (PEG-DAG) as described in U.S. Patent Publication Nos. 2003 / 0077829 and 2005 / 008689, PEG coupled to dimyristoylglycerol (PEG-DMG) as described in Abrams et al., Molecular Therapy 2010, 18(1), 171, PEG coupled to phosphatidylethanolamine (PE) (PEG-PE), or PEG conjugated to 1,2-di-O-hexadecyl-sn-glyceride (PEG-DSG), or mixtures thereof (see U.S. Patent No. 5,885,613). For example, the BSC can be a conjugated lipid that inhibits aggregation of lipid nanoparticles.

[0168] For example, the LNP can comprise a neutral lipid, e.g., a phospholipid or an analog or derivative thereof, such as a structured lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof, or a PEG-lipid, e.g., selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0169] The truncated human midkine protein, polynucleotide encoding the truncated human midkine protein, or SSO of the present disclosure can be conjugated to one or more moieties or groups that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide or protein. These moieties or groups can be covalently attached to functional groups such as primary or secondary hydroxyl groups. Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, peptides, N-acetylgalactosamine, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. The SSO of the present disclosure can be formulated with one or more pharmaceutically acceptable carriers, diluents, or excipients to aid administration. Alternatively, the truncated human midkine protein, polynucleotide encoding the truncated human midkine protein, or SSO of the present disclosure can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0170] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and / or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0171] In general terms, "carrier, diluent, or excipient" refers to a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any mammal, e.g., a human. Depending on the particular route of administration, various acceptable carriers, diluents, or excipients known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991). Pharmaceutically acceptable carriers can be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricating agents, adjuvants, vehicles, emulsifiers, absorbing agents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, sequestering agents, isotonic agents, and absorption delaying agents that do not affect the activity of the active agents of the present disclosure.

[0172] In one example, the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2 to 7.6. In one example, the salt is a sodium salt or a potassium salt.

[0173] A truncated human midkine protein, a polynucleotide encoding the truncated human midkine protein, or an SSO may contain a chiral (asymmetric) center, or the molecule as a whole may be chiral. Individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are within the scope of the present disclosure.

[0174] The compositions of the present disclosure can be formulated as pharmaceutically acceptable salts, esters, or salts of esters, or any other compounds that can provide (directly or indirectly) biologically active metabolites upon administration. As used herein, the term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of a composition that retain the desired biological activity of the parent compound and do not impart undesired toxic effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0175] In one example, the truncated human midkine protein, the polynucleotide encoding the truncated human midkine protein, or the SSO of the present disclosure can be complexed with a complexing agent to increase its cellular uptake. Examples of complexing agents include cationic lipids. The cationic lipids can be used to deliver the composition into cells.

[0176] The term "cationic lipid" includes lipids and synthetic lipids that have both polar and nonpolar domains, can be positively charged at or near physiological pH, and bind polyanions, such as nucleic acids, facilitating their delivery into cells. Cationic lipids generally include saturated and unsaturated alkyl and alicyclic ethers, as well as esters of amines, amides, or their derivatives. The linear and branched alkyl and alkenyl groups of cationic lipids can contain, for example, from 1 to about 25 carbon atoms. Preferred linear or branched alkyl or alkene groups have 6 or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions), including, for example, Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

[0177] Examples of cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, Lipofectamine™ (e.g., Lipofectamine™ 3000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3.beta.-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The oligonucleotides and proteins can also be complexed with, for example, poly(L-lysine) or avidin, and lipids may or may not be included in the mixture, for example, steryl-poly(L-lysine).

[0178] Cationic lipids have been used in the art to deliver oligonucleotides and proteins to cells (see, for example, US 5,855,910, US 5,851,548, US 5,830,430, US 5,780,053, US 5,767,099, US 10583201, US 10912833, EP 15797506.1, Lewis et al., 1996, Hope et al., 1998). Other lipid compositions that can be used to facilitate the uptake of the composition can be used in conjunction with the method of the present disclosure. In addition to those listed above, other lipid compositions are also known in the art, including, for example, those taught in US 4,235,871, US 4,501,728, US 4,837,028, US 4,737,323.

[0179] In one example, the lipid composition can further include agents, such as viral proteins, to enhance lipid-mediated transfection of oligonucleotides and proteins. In another example, N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides and proteins.

[0180] In another example, compositions of the present disclosure for delivery can include peptides having about one to about four basic residues. These basic residues can be located, for example, at the amino terminus, C-terminus, or internal regions of the peptide. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (which can also be considered nonpolar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, most or all of the other residues of the peptide can be selected from non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. In certain instances, a preponderance of neutral amino acids with long neutral side chains is used.

[0181] In one example, a truncated human midkine protein, a polynucleotide encoding a truncated human midkine protein, or an SSO of the present disclosure is modified by attaching a peptide sequence that assists in the transport of an oligonucleotide or protein into a cell, referred to herein as a "transport peptide" or "cell-penetrating peptide (CPP)." In one example, a truncated human midkine protein, a polynucleotide encoding a truncated human midkine protein, or an SSO of the present disclosure is covalently linked to a transport peptide or CPP.

[0182] As described herein, the truncated midkine protein of the present disclosure, the polynucleotide encoding it, or the SSO can be contained and provided within exosomes.

[0183] As used herein, the term "exosome" refers to small (20-300 nm diameter, more preferably 40-200 nm diameter) cell-derived vesicles that contain a membrane surrounding an internal space and are generated from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes are a type of extracellular vesicle. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes are derived from producer cells and can be isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0184] The truncated human midkine protein of the present disclosure or a polynucleotide encoding the truncated human midkine protein can be loaded into exosomes or engineered exosomes (see, for example, Olmeda D et al (2017) Nature 546, 676-680). Exosomes may contain a truncated protein or polynucleotide that acts on a target (e.g., a target cell) that is in contact with the exosome. After release from exosome-producing cells, exosomes can be delivered to target cells (i.e., recipient cells), where the exosomes are internalized and the exosomal cargo (e.g., a truncated human midkine protein of the present disclosure or a polynucleotide encoding the truncated human midkine protein) is delivered to the cytoplasm of the target cell.

[0185] Alternatively, when such exosomes are delivered to target cells, the exosome cargo can be delivered to the cytosol of target tissue cells through plasma membrane fusion. Such exosomes containing exosome cargo can be used for the treatment of various diseases in vivo. For example, exosomes containing a truncated human midkine protein or a polynucleotide encoding the same can be prepared and then delivered to target cells. In other words, exosomes can be used as drugs, and exosomes work better than conventional liposomes.

[0186] In vitro functional assays Various in vitro assays are available to evaluate the ability of a truncated human midkine protein, a polynucleotide encoding a truncated human midkine protein, or an SSO of the present disclosure to inhibit the interaction between human midkine and its ligand and / or midkine activity, and / or to treat a midkine-related disease or condition.

[0187] For example, a "scratch-wound assay" can be used to assess the ability of a truncated midkine protein of the present disclosure to inhibit the interaction between human midkine and its ligand and / or to inhibit human midkine activity. Such assays are based on the functional migration of cells in vitro or in vivo. Cell migration can be assessed by any suitable means, for example, in assays utilizing 96-well plates, or using other art-recognized methods for assessing cell migration.

[0188] In yet another example, chemotaxis assays can be used to evaluate the ability of the truncated midkine proteins of the present disclosure to inhibit the interaction between human midkine and its ligand and / or to inhibit human midkine activity. These assays are based on the functional migration of cells in vitro or in vivo induced by a compound (chemoattractant). Chemotaxis can be assessed by any suitable means, for example, in assays utilizing 96-well chemotaxis plates or using other art-recognized methods for assessing chemotaxis.

[0189] Generally, chemotaxis assays monitor the directional movement or migration of suitable cells within or through a barrier (e.g., endothelium, filter) from a first surface of the barrier to an opposing second surface toward an increasing level of a compound. Membranes or filters provide convenient barriers such that the directional movement or migration of suitable cells within or through the filter from a first surface of the filter to an opposing second surface of the filter toward an increasing level of a compound is monitored. In some assays, the membrane is coated with a substance to promote adhesion, such as ICAM-1, fibronectin, or collagen.

[0190] For example, the inhibition of migration of cells in a suitable container (containing means) from a first chamber into or through a microporous membrane to a second chamber can be detected or measured. The second chamber contains a chemoattractant, e.g., midkine protein, and a truncated midkine protein to be tested, and is separated from the first chamber by a membrane. Suitable membranes are selected with pore sizes suitable for monitoring specific migration in response to compounds, including, for example, nitrocellulose and polycarbonate. For example, pore sizes of about 3 to 8 microns, and preferably about 5 to 8 microns, can be used. The pore size can be uniform across the filter or within a suitable pore size range.

[0191] To assess migration and inhibition of migration, the distance of migration into the filter, the number of cells that traverse the filter and remain attached to the second surface of the filter, and / or the number of cells that accumulate in the second chamber can be determined using standard techniques (e.g., microscopy and flow cytometry). In one embodiment, cells are labeled with a detectable label (e.g., a radioisotope, fluorescent label, antigen, or epitope label), and migration can be assessed in the presence and absence of truncated midkine protein by determining the presence of the label attached to the membrane and / or present in the second chamber using an appropriate method (e.g., by detecting radioactivity, fluorescence, or immunoassay). The extent of induced or inhibited migration can be determined relative to a suitable control (e.g., compared to background migration determined in the absence of truncated midkine protein, compared to the extent of migration induced by the second compound (i.e., a standard), or compared to the migration of untransfected cells induced by the truncated midkine protein).

[0192] In one embodiment, a population of cells capable of binding to or migrating to midkine protein, e.g., a population of UMR106 cells, is placed in a chamber of a cell culture device that is in fluid communication with another chamber containing midkine protein (a chemoattractant). The two chambers are separated by a suitable membrane, e.g., a membrane that mimics the extracellular matrix found in a subject. The amount of cell migration through the membrane from one chamber to the other is assessed in the presence or absence of truncated midkine protein. A truncated midkine protein that prevents or reduces the amount of midkine-mediated cell migration compared to a control sample (containing no truncated midkine protein) is considered to have midkine inhibitory activity.

[0193] An exemplary assay for assessing the ability of the truncated midkine proteins described herein to bind to midkine protein is the cell migration assay described, for example, in Example 3 herein and in Martinotti S and Ranzato E 2020, Scratch wound healing assay. Methods Mol Biol 2109:225, which is incorporated herein by reference.

[0194] In another example, cell viability assays can be used to evaluate the ability of the truncated midkine proteins of the present disclosure on cell proliferation. These assays are based on the functional direct cytotoxic effect or cell death induced by the truncated midkine proteins in vitro or in vivo. Cell viability can be assessed by any suitable means, for example, in assays utilizing multiwell plates or using other art-recognized methods for assessing cell viability (see, e.g., Riss et al. 2013, Assay Guidance Manual, Cell Viability Assays).

[0195] As will be apparent to one of skill in the art, screening methods may involve detecting levels of cell death, cell proliferation, and / or cell survival. Such methods are known in the art.

[0196] In vivo functional assays In another example, the effectiveness of the truncated human midkine of the present disclosure for inhibiting human midkine activity and / or treating a disease or condition is assessed using an in vivo assay.

[0197] For example, a truncated human midkine of the present disclosure can be administered to a non-human mammal (e.g., mouse) model of cancer. A truncated human midkine that reduces or alleviates at least one symptom associated with cancer, e.g., tumor size or volume, metastasis, in a mammalian subject relative to the cancer or its symptoms in the subject prior to administration and / or relative to the cancer or its symptoms in a control mammal to which the truncated human midkine has not been administered, is considered suitable for treating the disease or condition.

[0198] Dosage and Regimen For the prevention or treatment of a disease or condition or its recurrence, the appropriate dosage of an active agent (e.g., a truncated human midkine protein of the present disclosure, a polynucleotide encoding a truncated human midkine protein, or an SSO) will depend on the type of disease being treated, the severity and course of the disease, whether the active agent is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the active agent, and the discretion of the attending physician. Typically, a therapeutically effective amount of the composition will be administered. The particular dosage regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history as evaluated by the physician. Typically, the clinician will administer the active agent until a dosage is reached that achieves the desired result.

[0199] As used herein, the terms "treating," "treat," or "treatment" include administering a therapeutically effective amount of a compound described herein sufficient to reduce or eliminate at least one symptom of a disease, disorder, or condition.

[0200] As used herein, the terms "preventing," "prevent," or "prevention" include administering a therapeutically effective amount of a compound described herein sufficient to arrest or hinder the onset of at least one symptom of a disease, disorder, or condition.

[0201] The terms "therapeutically effective amount" and "effective amount" describe an amount of a particular agent, such as an oligonucleotide or protein of the present disclosure, sufficient to achieve a desired effect in a subject or cell treated or contacted with the agent. For example, this can be the amount of a composition including one or more agents that inhibit the activity of one or more nucleic acid sensors described herein necessary to reduce, alleviate, and / or prevent a disease, disorder, or condition. In some examples, a "therapeutically effective amount" is sufficient to reduce or eliminate symptoms of a disease, disorder, or condition. In another example, a "therapeutically effective amount" or "effective amount" is an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce or prevent an aging-related disease, disorder, or condition in a cell, or to inhibit or prevent aging in a cell.

[0202] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantial cytotoxic effects in the subject. The effective amount of an agent useful for reducing, alleviating, and / or preventing a disease, disorder, or condition will depend on the subject being treated, the type and severity of any associated symptoms, and the manner of administration of the therapeutic composition.

[0203] Generally, dosages vary depending on the patient's age, condition, sex, and severity of the disease, and can be determined by those skilled in the art. Dosages can be adjusted by individual physicians in the event of any complications. For in vivo administration of the compositions described herein, typical dosage amounts can range from about 10 ng / kg to about 100 mg / kg or more of an individual's body weight per day. Exemplary dosage amounts and ranges are described herein. For repeated administration over several days or longer, depending on the severity of the disease or disorder being treated, treatment can be sustained until the desired suppression of symptoms is achieved.

[0204] The dosage for a particular composition can be empirically determined in a mammal that has received one or more administrations of each composition. To evaluate the effectiveness of the compositions of the present disclosure, the clinical symptoms of the composition or the condition being treated, such as cancer, can be monitored. For example, the effectiveness of the SSO of the present disclosure in treating cancer can be evaluated based on tumor size and / or using cancer diagnostic, prognostic, or predictive biomarkers.

[0205] Administration of the compositions according to the disclosed methods can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the compositions can be essentially continuous over a preselected period of time, or can be in a series of spaced doses.

[0206] Various routes of administration may include, but are not necessarily limited to, oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g., intrabronchial, intraocular, intranasal, or oral inhalation, intranasal instillation), depending on the disease or condition being treated. Other suitable methods of administration may also include rechargeable or biodegradable devices, and slow-release biological or synthetic polymeric devices.

[0207] use The compositions of the present disclosure have the ability to inhibit midkine function and therefore can be used as therapeutic and preventive drugs for midkine-related diseases and disorders.

[0208] In one example, the present disclosure provides a method for inhibiting the interaction between human midkine and its ligand on the surface of or within a cell, the method comprising exposing the cell to a composition described herein. It will be understood by those skilled in the art that the method can be performed in vitro, ex vivo, or in vivo.

[0209] The cell can be any known in the art to express midkine. By way of example, the cell can be an immune cell, such as a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, and a dendritic cell; an endothelial cell; or a neuron. In oncology, the cell can be a malignant tumor cell or a stromal cell, such as a fibroblast or an endothelial cell.

[0210] In another example, the present disclosure provides a method for inhibiting human midkine activity in a cell, the method comprising exposing the cell to a composition described herein.

[0211] As used herein, the term "inhibit" shall be taken to mean blocking, reducing, suppressing, or preventing midkine activity in a cell relative to midkine activity in a cell in which a truncated human midkine protein of the present disclosure or a polynucleotide encoding a truncated human midkine protein is present, or in which an SSO of the present disclosure is absent.

[0212] Midkine activity can be inhibited by any measurable amount. Inhibition of midkine activity can be complete or partial. Thus, the methods disclosed herein can include at least partial inhibition of midkine activity. For example, midkine activity can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% after contacting a cell with an effective amount of a composition (e.g., relative to the same measured value of activity before contact with the composition).

[0213] In another example, the present disclosure provides a method for treating or preventing a midkine-associated disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition described herein.

[0214] In yet another example, the present disclosure provides use of a composition described herein in the preparation of a medicament for the treatment or prevention of a midkine-associated disease or disorder.

[0215] The term "midkine-associated disease" refers to a disease involving midkine function. Examples of such diseases include cancer (esophageal cancer, thyroid cancer, bladder cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, prostate cancer, and Wilms' tumor) and diseases caused by cell growth or angiogenesis, such as endometriosis; inflammatory diseases, or diseases caused by recruitment of inflammatory cells, suppression of regulatory T cell function, activation of neutrophils, or dysfunctional orientation of macrophages or T cells, such as renal inflammatory diseases, acute renal failure, chronic kidney disease, osteoporosis, sepsis, arthritis, autoimmune diseases (organ-specific autoimmune diseases, etc.), rheumatoid arthritis (rheumatoid arthritis (RA) or osteoarthritis (OA)), multiple sclerosis (relapsing-remitting multiple sclerosis, etc.), inflammatory bowel diseases (Crohn's disease, etc.), These diseases include systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Sjögren's syndrome, polymyositis (PM), dermatomyositis (DM), polyarteritis nodosa (PN), thyroid diseases (such as Graves' disease), Guillain-Barré syndrome, primary biliary cirrhosis (PBC), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, experimental autoimmune myasthenia gravis (EAMG), amyotrophic lateral sclerosis (ALS), type 1 diabetes mellitus, transplant rejection, postoperative adhesions, endometriosis, psoriasis, lupus, allergies, asthma, acute respiratory distress syndrome; ventilation-induced lung injury; and diseases caused by occlusive vascular disease or vascular intimal hyperplasia, such as post-vascular revascularization restenosis, coronary artery occlusive disease, cerebrovascular occlusive disease, renal vascular occlusive disease, peripheral occlusive disease, arteriosclerosis, and cerebral infarction. In one example, the midkine-associated disease or disorder is selected from an autoimmune disease, cancer, or inflammatory disease.

[0216] In certain examples, diseases treated or prevented using the compositions of the present disclosure exhibit increased, excessive, or abnormal midkine expression, accumulation, activity, and / or signaling. Such diseases include, but are not limited to, those described herein. In one example, the compositions of the present disclosure can be used in a method for preventing or inhibiting inflammation associated with the administration of a therapeutic SSO, such as those known in the art, to a subject. In particular, the SSOs described herein can be used in the prevention or inhibition of inflammation mediated by one or more nucleic acid sensors (e.g., TLR3, TLR7, TLR8, TLR9, cGAS, RIG-I) during or after the administration of the therapeutic SSO. It is contemplated that inflammation can involve or include any cell, tissue, or organ of the body. In certain examples, inflammation is or includes liver inflammation. To this end, therapeutic SSOs can be conjugated to N-acetylgalactosamine (GalNAc), which enhances asialoglycoprotein receptor (ASGR)-mediated uptake into liver hepatocytes (Nair et al., 2014), thereby enabling their specific targeting to the liver.

[0217] In certain instances, therapeutically effective amounts of therapeutic SSOs and compositions of the present disclosure may be administered simultaneously, concurrently, sequentially, consecutively, alternatingly, or separately in any particular combination and / or order.

[0218] According to the above-mentioned uses, the composition of the present disclosure can be administered to an animal. For example, the animal (or subject) to which the composition is administered can be a mammal, a bird, a chordate, an amphibian, or a reptile. In one example, the animal is a mammal. Exemplary mammalian subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). In one example, the mammal is a human.

[0219] Combination therapy The compositions of the present disclosure can also be administered as part of a combination therapy with other agents useful for treating a disease or condition, such as cancer, either as a combined or additional therapeutic step, or as an additional component of a therapeutic formulation. Such other therapies / agents will be known to those of skill in the art.

[0220] For example, the other compound is an anti-inflammatory or immunomodulatory drug. Alternatively, or additionally, the other compound is an immunosuppressant. Alternatively, or additionally, the other compound is a chemotherapeutic agent, such as carboplatin.

[0221] In order that preferred embodiments of the present disclosure may be fully understood and put to practical use, reference is made to the following non-limiting examples. [Table 1] [Table 2-1] [Table 2-2] [Table 3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Example]

[0222] Example 1: Primer design and optimization There are nine protein-coding MDK transcripts reported on Ensembl. The MDK-203 transcript (NM_002391.6) was used as the basis for exon numbering. The majority of transcripts differ only by their 5' untranslated regions, but transcripts 204, 209, 207, and 213 also have variations within the coding region (see Figure 2).

[0223] Five primers were designed to amplify the MDK region of interest. The sequences and expected polymerase chain reaction (PCR) product sizes of the various primer sets are outlined in Table 5. The binding position of each primer on the MDK genomic sequence is shown in Figure 3. [Table 5]

[0224] Six primer sets were optimized by modifying the PCR method, annealing temperature, number of cycles, and cell type (see Figure 4). The optimized amplification conditions were specified as a TaKaRa La Taq PCR system using 2x GC buffer I and the following thermal cycling conditions: 94°C for 1 minute, followed by 25–27 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 2 minutes. The primer set containing MDK Ex1F should amplify transcripts 203, 204, and 213, while all other primer sets should amplify all transcripts. When using the optimized TaKaRa PCR protocol, each primer set amplifies a single product of approximately the expected size for a transcript containing all four coding exons.

[0225] Example 2: Design and synthesis of SSO The first-generation MDK SSOs are shown in Table 6. Factors considered in the design strategy included optimal overlap with splice enhancer sequence motifs identified using Splice Aid software, while aiming to minimize G-blocks in the antisense SSO, biased G+C composition, and runs of more than 3C to avoid secondary structures in the mRNA. After screening the generation 1 SSO sequences, additional SSOs were created by "microwalking" around the effective sequences. Microwalking refers to moving the target sequence upstream or downstream of the original sequence. In this case, second-generation SSOs were designed by shifting 5 or 10 bases in each direction of the first-generation sequence that induced exon skipping (see Table 6). A 20-mer version of the most effective exon 3 SSO was also ordered. All SSOs were ordered from SynGenis (WA, Australia) as 2'-O-methyl-modified bases (2'OMe-PS) on a phosphorothioate backbone. The target sequences of these SSOs relative to splicing factor binding site motifs predicted using SpliceAid1 are shown in Figure 5 . [Table 6]

[0226] Example 3: 2'OMe-PS SSO screening in Huh7 Early SSO screening in Huh7 The human hepatoma-derived HuH-7 cell line (Huh7) was chosen for initial SSO screening due to its reported expression of MDK and ease of transfection. Huh7 cells were transfected with 2'OMe-PS SSO using Lipofectamine 3000 at three concentrations: 200 nM, 100 nM, and 50 nM. Cells were lysed and harvested for RNA extraction and analysis after a 24-hour incubation period.

[0227] This initial SSO screen revealed that for both exon 3 and exon 4, the two SSOs closest to the donor splice sites were most effective at inducing exon skipping (see Figure 6). Exon 3 (Δ3) skipping was more efficient than exon 4 (Δ4), with approximately 32% Δ3 product after transfection with 3A (+143 +167) at 50 nM (see Figure 6a). The highest percentage of Δ4 product (11%) was achieved with 4A (+106 +130) at 50 nM (see Figure 6b). The exon 4 SSO also induced skipping of both exons 3 and 4 (Δ3 + 4), as well as an intermediate band representing cryptic splicing in some cases (see Figure 6b). The first two SSOs targeting exon 3 also yielded a small PCR product (FL + i2) with inclusion of intron 2.

[0228] Cocktails at Huh7 Huh7 cells were co-transfected with two 2'OMe-PS SSOs as a cocktail. It is often observed that two SSO sequences that are individually inefficient induce efficient skipping when used as a cocktail. Two concentrations, 100 nM and 50 nM, were evaluated. Each SSO was used in equimolar amounts within these total cocktail concentrations, e.g., 50 nM of each SSO for a total cocktail concentration of 100 nM. Cells were lysed and harvested 24 hours later for RNA extraction and analysis.

[0229] The SSO cocktails resulted in splicing patterns similar to those of the individual SSOs. The two SSOs closest to the donor splice site induced the most efficient skipping for exons 3 and 4. Although the concentration of each SSO was lower than those previously tested, the 50 nM total concentration treatment was largely the most effective and induced a higher percentage of skipped products. Combining 3A (+143 +167) with any other exon 3 SSO resulted in more than 40% Δ3 product (see Figure 7a). Most of the cocktails for exons 3 and 4 resulted in a small percentage of Δ3 +4 product, but these products were also observed in both GTC and UT samples, suggesting that this may be naturally occurring skipping (see Figure 7). The combination of 4A (+106 +130) and 4D (+10-15) was the most effective at inducing exon 4 skipping, resulting in 26% Δ4 product (see Figure 7b).

[0230] Lower concentrations in Huh7 Because previous screening experiments showed that concentrations as low as 50 nM were effective, most effective for the exon 3 SSO, a titration of lower concentrations was evaluated. Huh7 cells were transfected with 2'OMe-PS SSO using Lipofectamine 3000 at three concentrations: 50 nM, 25 nM, and 12.5 nM. Cells were lysed and harvested for RNA extraction and analysis 24 hours after transfection.

[0231] Both sequences, 3A (+143+167) and 3D (+15-10), induced exon skipping in a dose-dependent manner up to 12.5 nM, with 50 nM producing the most exon 3 skipping (see Figure 8a). Exon 4 skipping was also dose-dependent, with 50 nM inducing the highest proportion of Δ4 product, although this skipping was less effective than exon 3, with a maximum of 10% Δ4 product (see Figure 8b).

[0232] Microwalking SSO in Huh7 Second-generation MDK SSOs were designed by "microwalking" around the effective generation 1 SSO sequence. Microwalking refers to moving the target sequence upstream or downstream of the original sequence. In this case, the second-generation SSO was designed 5 or 10 bases in each direction of the first-generation sequence that induced exon skipping (Table 6). A 20-mer version of the most effective exon 3 SSO was also synthesized. All SSOs were ordered from SynGenis (WA, Australia) as 2'-O-methyl-modified bases on a phosphorothioate backbone (2'OMe-PS). Huh7 cells were transfected with the microwalked and corresponding original 2'OMe-PS SSOs using Lipofectamine 3000 at two concentrations: 100 nM and 50 nM. Cells were lysed and harvested 24 h posttransfection for RNA extraction and analysis.

[0233] Exon 3 SSOs microwalked from 3A(+143+167) toward the acceptor site were less effective than the original sequence, as were 20-mer sequences (see Figure 9a). In contrast, the new 3D(+05-20) sequence effectively induced up to 45% exon 3 skipping (see Figure 9a). SSOs microwalked around the original 4A(106+130) showed an interesting phenomenon: on the one hand, 4A(+111+145) induced more cryptic splicing and less exon 4 skipping than the original, while on the other hand, 4A(+101+125) induced more exon 4 skipping without causing cryptic splicing (see Figure 9b). Similar results were seen with sequences microwalked around 4A(+74+98), where 4A(+69+93) induces multiple nonspecific skipped products and 4A(+79+103) induces more specific exon 4 skipping (see Figure 9b).

[0234] Exon 3+4 cocktail in Huh7 Huh7 cells were co-transfected with two 2'OMe-PS SSOs as a cocktail. However, each cocktail consisted of one SSO for each of exon 3 and exon 4. The goal was to minimize the proportion of full-length product. Two concentrations were evaluated: 100 nM and 50 nM. Each SSO was used in equimolar amounts within these total cocktail concentrations, e.g., 50 nM of each SSO for a total cocktail concentration of 100 nM. Cells were lysed and harvested 24 hours later for RNA extraction and analysis.

[0235] Treatment with the exon 3 + 4 cocktail resulted in the expected splicing pattern, with those cocktails containing effective SSOs for either exon 3 or exon 4 inducing Δ3 + 4 skipping (see Figure 10). The exception to this was the combination of 3A(+143+167) or 3D(+15-10) with either of the two SSOs targeting the exon 4 acceptor site; these cocktails induced only single-exon skipping, likely exon 3 (see Figure 10). Neither cocktail was as effective as the 3A(+143+167) SSO used as a positive control, which reduced the percentage of FL product to 58%. The combination of 3A(+143+167) and 4D(+10-15) was the most effective cocktail, reducing the percentage of FL product to approximately 60% at 100 nM (see Figure 10).

[0236] Example 4: 2'OMe-PS SSO screening in SHSY5Y The 2'OMe-PS SSO screen performed in Huh7 cells was repeated in the neuroblastoma-derived SHSY5Y cell line. SHSY5Y cells were transfected with either individual SSOs at 200, 100, and 50 nM, or a cocktail of the two SSOs at 100 and 50 nM. Cells were lysed and harvested 24 hours later for RNA extraction and analysis.

[0237] After transfection of SHSY5Y cells with individual SSOs, the splicing patterns were similar to those observed in Huh7 cells. The 3A (+143 +167) and 3D (+15-10) SSOs induced the highest proportion of Δ3 products, up to 37% and 23%, respectively. Interestingly, the strong dose response observed in Huh7 cells was not seen in SHSY5Y cells at concentrations of 200 nM and 50 nM, resulting in similar proportions of exon-skipped products (see Figure 11a). The FL+i2 product was also more prominent in SHSY5Y cells, accounting for more than 5% of PCR products at all concentrations after transfection with either of the SSOs covering the acceptor splice site (see Figure 11a). Exon 4 skipping using individual SSOs was similarly inefficient in both cell lines, with both producing only minimal amounts of the desired Δ4 product, as well as Δ3+4 and cryptic products (see Figure 11b).

[0238] While the splicing product patterns after transfection with the two SSO cocktails are similar between Huh7 and SHSY5Y cells, the proportion of skipped products is much lower in SHSY5Y cells (see Figure 11c). We also note that the higher 200 nM concentration is more effective in SHSY5Y cells, while the lower 50 nM concentration is most effective in Huh7 cells. This reduction in efficiency may be due to the increase in the number of cells used per transfection, from 50,000 in Huh7 to 75,000 in SHSY5Y. However, it may also be a result of lower transfection efficiency in SHSY5Y cells compared to Huh7 cells.

[0239] Example 5: 2'OMe-PS SSO screening in HMC-1 Initial transfection of promising AO in HMC-1 A human mast cell line (HMC-1) was transfected with the most promising SSO for each MDK exon using Lipofectamine 3000. The transfection protocol was modified because these cells had not been previously transfected and were suspension cells. HMC-1 cells were transfected at 6 × 10 5 The cells were transferred to 24-well plates at a density of 1000 cells / ml. On the day of transfection, the delivery complex containing SSO and Lipofectamine 3000 diluted in 50 μl of OptiMEM, as recommended by the manufacturer, was incubated for 15 minutes and then added directly to the cells in growth medium. This protocol differs from that used for adherent cell lines, in which cells are similarly seeded 24 hours before transfection. However, after the 15-minute incubation, the delivery complex is further diluted in OptiMEM and added to the cells, replacing the cell growth medium. HMC-1 cells were lysed and harvested for RNA extraction and analysis 24 hours after transfection.

[0240] Both the single SSO targeting exon 3 and the two-SSO cocktail effectively induced exon skipping (see Figure 12a). In contrast, no exon 4 skipping was observed (see Figure 12a). The SMN positive control revealed poor transfection efficiency, with only approximately 50% SMN exon 7 skipping (see Figure 12b). With efficient transfection, we would expect to see 80-100% SMN skipping. Additionally, the spontaneous exon 7 skipping seen in untreated samples is often observed in our laboratory when cells are stressed.

[0241] To prevent contaminating gDNA, RNA samples were treated with ezDNAse prior to cDNA synthesis and PCR amplification. Despite the effectiveness of ezDNAse treatment (Figure 12c), larger PCR products with sizes roughly corresponding to genomic DNA were still present, suggesting that unprocessed transcripts are retained in HMC-1 mast cells.

[0242] Second transfection with fewer cells Transfection of HMC-1 cells was performed using 6 × 10 6 rather than 1 × 10 per treatment. 6 The experiment was repeated using 100 cells. Despite the increased efficiency indicated by 79% SMN exon 7 skipping (see Figure 13b), only a slight increase in skipping efficiency was observed, and exon 4 SSO was ineffective (see Figure 13a).

[0243] Microwalking exon 4 SSO in HMC-1 cells Two microwalking SSOs for exon 4 (see Figure 9b), which were identified as more effective than the original sequence, were transfected into HMC-1 cells. Despite the poor transfection efficiency exhibited by the 3A (+143 +167) positive control SSO, the microwalking exon 4 SSO successfully induced exon 4 skipping (see Figure 14). To increase the transfection efficiency of HMC-1 cells, a more suspension cell line-friendly transfection method, such as electroporation, may be required.

[0244] Example 6: PMO Evaluation The most promising SSO sequences for each exon were ordered as phosphorodiamidate morpholino oligomer (PMO) chemistry (Table 6). The selection of SSO sequences for PMO synthesis from each exon was based on the results of previous experiments conducted in HuH7, SHSY5Y, and HMC-1 cells as outlined above and confirmed in final testing in HuH7 cells (Figure 15). This identified 3A (+143 +167), 4A (+79 +103), and 4A (+101 +125) as the best candidates for PMO evaluation, while 3D (+21-04) was not developed as a PMO SSO due to unfavorable characteristics of the RNA sequence surrounding this region of exon 3.

[0245] One additional sequence, MDK 4A (+100 +124), was also designed due to concerns from Gene Tools that the percentage of G nucleotides (40%) in the MDK 4A (+101 +125) sequence exceeded their recommended upper limit (36%). Note that the MDK H4A (+79 +103) sequence was also flagged for high self-complementarity, which may lead to dimer formation and lower antisense activity. SHSY5Y cells were transfected with PMO using the Neon electroporation transfection method. Three concentrations were evaluated: 20 μM, 10 μM, and 5 μM. These concentrations were calculated in a 10 μl Neon chip and roughly correlate to 200 nM, 100 nM, and 50 nM in 1 ml. Cells were harvested 24 h posttransfection due to concerns about cell proliferation and the rate of cell death (approximately 50%), which could lead to dilution of the PMO effect. The PMO SSO directed against exon 3 reached a skipping efficiency of approximately 80%, while the PMO SSO directed against exon 4 reached a skipping efficiency of approximately 40% (see Figure 16). [Table 7]

[0246] Example 7: Western blot evaluation Huh7 and SHSY5Y cells were transfected with the most promising exon 3 and 4 AOs, both individually and as a two-AO cocktail, using Lipofectamine 3000. Cells were incubated for 48 hours after transfection and then harvested for RNA or protein analysis. RNA analysis revealed that transfection was efficient at inducing skipping of both exon 3 and exon 4 (Figure 17, upper panel). Western blotting clearly shows a reduction in full-length midkine in the control lanes (GTC, ZAP, UTC) relative to the short forms in all tracks with the exon 4 SSO (Figure 17, lower panel).

[0247] FIG. 17 provides evidence for the production of a truncated human midkine protein with an exon 4 deletion.

[0248] Example 8: Midkine mRNA Midkine mRNA containing the full-length midkine coding region (SEQ ID NO: 96), including the 5' UTR; cap structure; 3' UTR, and the natural initiation codon and peptide leader sequence linked to a poly AAA sequence, was synthesized by in vitro transcription using a standard plasmid template and RNA polymerase.

[0249] Deletion mRNAs were designed to encode truncated midkine proteins lacking 14, 25, 34, 41, and 58 amino acids from the C-terminus (shown in SEQ ID NOs: 86, 88, 90, 92, and 94, respectively). The deletion mRNAs contained the same 5' UTR, cap structure, 3' UTR, and poly-AAA sequence as those used for the full-length midkine sequence.

[0250] A deletion mRNA encoding a truncated midkine protein lacking 41 amino acids from the C-terminus (shown in SEQ ID NO: 92) was included in the experiment as a comparative substance, as reported in the literature (Dianat et al. 2014). Although Dianat et al. 2014 refers to a variant lacking the last 40 amino acids, those skilled in the art will understand from the name MDKΔ81-121 mentioned in the paper that midkine actually lacks the last 41 amino acids.

[0251] Example 9: Detection of truncated midkine protein produced by midkine mRNA deletion in cancer cells Transfection of breast cancer cells with midkine mRNA: 2,000 MDA-MB-231 breast cancer cells per well were plated in 1 mL of DMEM and 10% FBS in a 96-well plate. Cells were incubated overnight at 37°C in 5% CO2.

[0252] Transfection medium was prepared by combining 1 μL of Lipofectamine MessengerMax (catalog no. LMRNA001), 0.2 ng / 1000 cells of midkine deletion mRNA template (each well received one of the deletion mRNA sequences shown in SEQ ID NOs: 86, 88, 90, 92, 94, and 96), and Opti-MEM to a final volume of 50 μL.

[0253] Cells were incubated in transfection medium at 37°C in 5% CO for 5-6 hours, then 150 μL of DMEM and 10% FBS was added to each well containing 50 μL of Opti-MEM, and incubation was continued for an additional 72 hours. Cell viability was assessed by adding Alamar Blue to the plate and capturing absorbance at 570-595 nm after 13 hours.

[0254] Midkine mRNA deletions Δ14, Δ25, Δ34, and Δ58 all showed reduced cancer cell viability compared with full-length midkine mRNA, indicating that truncated midkine lacking 14, 25, 34, and 58 amino acids from the C-terminus interferes with breast cancer cell proliferation (see Figure 21). Furthermore, it should be noted that midkine mRNA deletion Δ41 is equivalent to full-length midkine and is therefore not a suitable candidate.

[0255] Western blotting 18 μL of conditioned medium was loaded onto a precast 4-15% gradient Bio-Rad gel along with protein loading buffer. Proteins were separated and then immobilized on a membrane using a Bio-Rad transfer system.

[0256] The membrane was incubated overnight with MDK antibody (Invitrogen PA5-109951) at 1:1000, followed by a 1-hour incubation with secondary rabbit antibody at 1:5000 in 5% skim milk blocking buffer. The antibody was diluted in 2.5% skim milk. Detection of midkine-specific bands was performed using chemiluminescence.

[0257] The gradual reduction in band size from full-length midkine to Δ14 and finally Δ58 indicates the expected size when 14 to 58 amino acids are removed from the C-terminus of the truncated midkine protein (see Figure 19).

[0258] Example 10: Migration of breast cancer cells is reduced by midkine mRNA with 14 or 58 amino acid deletions from the C-terminus. Cell migration assay 50,000 MDA-MB-231 breast cancer cells were plated per well in 100 μL of DMEM and 10% FBS in a 96-well plate. The cells were incubated overnight in an incubator at 37°C in 5% CO2.

[0259] Transfection medium was prepared by combining Lipofectamine MessengerMax with mRNA encoding the Δ14 or Δ58 deletion variants at a concentration of approximately 1 ng of mRNA per 1000 cells. Cells were transfected in 50 μL of Opti-MEM.

[0260] Cells were incubated at 37°C in the presence of 5% CO2 for 5-6 hours, after which 150 μL of DMEM + 10% FBS was added to each well containing 50 μL of Opti-MEM. The medium was removed 24 hours after transfection, and 100 μL of PBS was then added. The semi-confluent monolayer of cells was then wound using the WoundMaker System, followed by one PBS wash. 150 μL of transfection medium was returned to each well. The plate was then placed in an IncuCyte and imaged every hour for 12 hours to determine cell migration into the gap. The area under the curve was calculated by quantifying the wound width at 1-hour intervals (Table 7).

[0261] Midkine mRNA deletion variants were compared with control green fluorescent protein (GFP) mRNA or full-length midkine mRNA. Midkine mRNAs Δ58 and Δ14 (SEQ ID NOs: 86 and 92, respectively) showed significant inhibition of breast cancer cell migration. Interestingly, however, midkine mRNA Δ41 had no effect on breast cancer cell migration. [Table 8]

[0262] Example 11: Proliferation of liver cancer cells is reduced by midkine-deficient mRNA. 10,000 Huh-7D12 cells were plated per well in 100 μL of DMEM and 10% FBS in a 96-well plate. The cells were incubated overnight at 37° C. in the presence of 5% CO 2 .

[0263] Transfection medium was prepared based on the initial cell density used in Example 9. Lipofectamine MessengerMax was added together with mRNA encoding one of the midkine deletion variants at a concentration of approximately 1 ng of mRNA per 1,000 cells. Cells were transfected in 50 μL of Opti-MEM. The cells were incubated at 37°C in the presence of 5% CO2 for 5-6 hours. 150 μL of DMEM and 10% FBS was added to each well containing 50 μL of Opti-MEM. 24 hours after transfection, the medium was removed, and the cells were washed once with PBS. Next, 150 μL of transfection medium was added back to each well, and the plate was placed in an incubator at 37°C in 5% CO2 for 72 hours. Alamar Blue was then added to the plate, and the absorbance was monitored at 570-595 nm after 5 hours to determine the number of viable cells.

[0264] Midkine mRNA deletion Δ58 reduced cell viability compared with full-length midkine mRNA, indicating that truncated midkine lacking 58 amino acids from the C-terminus interferes with liver cancer cell proliferation (see Figure 20).

[0265] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. 1. A composition comprising: (i) a truncated human midkine protein in which the amino acid sequence encoded by exon 4 of human midkine is absent, partially absent, or substantially absent, and / or (ii) A composition comprising a polynucleotide encoding the truncated human midkine protein in (i).

2. The composition of claim 1, wherein about 14, about 25, about 34, or about 58 contiguous amino acids at the C-terminus of human midkine protein are absent from the truncated human midkine protein.

3. The composition of claim 1 or 2, wherein the human midkine protein comprises the amino acid sequence shown in SEQ ID NO:

75.

4. The composition of any one of claims 1 to 3, wherein approximately 14 contiguous amino acids at the C-terminus of human midkine protein are absent from said truncated human midkine protein.

5. The composition of claim 4, wherein the truncated human midkine protein comprises or consists of the sequence shown in SEQ ID NO:

87.

6. The composition of any one of claims 1 to 3, wherein approximately 58 contiguous amino acids at the C-terminus of human midkine protein are absent from said truncated human midkine protein.

7. The composition of claim 4, wherein the truncated human midkine protein comprises or consists of the sequence shown in SEQ ID NO:

95.

8. The composition of any one of claims 1 to 7, wherein the polynucleotide is mRNA.

9. The composition according to claim 8, when added to any one of claims 1 to 5, wherein the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 14 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:

86.

10. 10. The composition of claim 9, wherein the polynucleotide comprises the sequence set forth in SEQ ID NO:

86.

11. 11. The composition of claim 9 or 10, wherein the polynucleotide sequence encodes the amino acid sequence shown in SEQ ID NO:

87.

12. The composition according to claim 8, when added to any one of claims 1 to 3 and 6 or 7, wherein the polynucleotide is an mRNA encoding a truncated human midkine protein in which approximately 58 contiguous amino acids at the C-terminus of the human midkine protein are absent, and the mRNA comprises or consists of a polynucleotide sequence having at least 80% identity to the sequence set forth in SEQ ID NO:

94.

13. The composition of claim 12, wherein the polynucleotide comprises the sequence set forth in SEQ ID NO:

94.

14. 14. The composition of claim 12 or 13, wherein the polynucleotide sequence encodes the amino acid sequence shown in SEQ ID NO:

95.

15. The composition of any one of claims 1 to 14, wherein the polynucleotide is codon-optimized.

16. The composition according to any one of claims 1 to 7, wherein the polynucleotide is a DNA sequence encoding the truncated midkine protein, and the polynucleotide is operably linked to a promoter and / or is contained in an expression vector.

17. The composition of any one of claims 1 to 15, wherein the polynucleotide is mRNA formulated with a lipid nanoparticle (LNP).

18. The composition of any one of claims 1 to 17, further comprising a pharmaceutically acceptable carrier or diluent.

19. A method for producing a truncated human midkine protein, comprising culturing cells containing the polynucleotide defined in claim 16 for a time and under conditions sufficient for the cells to produce the truncated human midkine protein, and optionally recovering the truncated human midkine protein from the culture.

19. A method for producing a truncated human midkine protein, comprising transfecting a cell with a polynucleotide defined in any one of claims 1 to 15 under conditions sufficient for the cell to produce the truncated human midkine protein, and optionally recovering the truncated human midkine protein from the culture.

20. A method for inhibiting the interaction between human midkine and its ligand on the surface of or within a cell, said method comprising exposing said cell to the composition of any one of claims 1 to 18.

21. The method of claim 20, wherein inhibition of the interaction between human midkine and its ligand reduces cancer cell viability and / or cell migration.

22. A method for inhibiting human midkine activity in a cell, said method comprising exposing said cell to the composition of any one of claims 1 to 18.

23. A method for treating or preventing a midkine-associated disease or disorder in a subject in need thereof, said method comprising administering to said subject the composition of any one of claims 1 to 18.

24. The method according to claim 23, wherein the midkine-associated disease or disorder is an autoimmune disease, cancer, an inflammatory disease, or multiple sclerosis.

25. The method according to claim 24, wherein the midkine-associated disease or disorder is cancer.

26. Use of the composition according to any one of claims 1 to 18 in the preparation of a medicament for treating or preventing a midkine-associated disease or disorder selected from autoimmune diseases, cancer, or inflammatory diseases in a subject in need thereof.

27. 27. The use according to claim 26, wherein the medicament is for the treatment of cancer.

28. Use of the composition according to any one of claims 1 to 18 for treating or preventing a midkine-associated disease or disorder selected from autoimmune diseases, cancer, or inflammatory diseases in a subject in need thereof.

29. The use according to claim 28, wherein the midkine-associated disease or disorder is cancer.

30. 30. The method of claim 25 or the use of claim 27 or 29, wherein the cancer is selected from the group consisting of esophageal cancer, thyroid cancer, bladder cancer, colorectal cancer, cutaneous and uveal melanoma, squamous cell carcinoma, osteosarcoma B-cell malignancies, leukemia, head and neck cancer, gallbladder cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, prostate cancer, and Wilms' tumor.

31. 30. The method of claim 25, or the use of claim 27 or 29, wherein the cancer is breast cancer.

32. 30. The method of claim 25, or the use of claim 27 or 29, wherein the cancer is liver cancer.

33. The method of any one of claims 23 to 24 and 30 to 32, and the use of any one of claims 26 to 32, wherein the subject is being treated or will be treated with a chemotherapeutic agent.

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