Compositions and methods for the treatment of kidney disease

Abstract

This specification describes antisense oligonucleotides, vectors, and related compositions and methods for increasing the expression of PKD1 mRNA and polycystin 1 protein, and their use for the treatment of autosomal dominant polycystic kidney disease (ADPKD).

Description

Technical Field

[0001] This application claims priority from Australian Patent Application No. 2023901907, filed on June 16, 2023, and Australian Patent Application No. 2023903042, filed on September 21, 2023, the entire contents of each of which are incorporated herein by reference. Technical Field The present disclosure generally relates to oligonucleotides, and related compositions and methods for treating conditions associated with mutations in the polycystic kidney disease 1 (PKD1) gene.

Background Art

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease leading to end-stage renal disease (ESRD). Approximately 1 in 500 - 2,500 people carry a mutation related to this condition. ADPKD is a progressive disorder characterized by abnormal expansion of renal tubular cells resulting in the growth of numerous cysts in the kidneys.

[0003] The development of cysts begins at an early stage of life, starting in utero in more severe cases, usually followed by a long asymptomatic progression period. The signs and symptoms of ADPKD typically appear between the ages of 30 and 40. However, approximately 3% of ADPKD patients have very early onset or an unusually rapid progressive disease. As renal function declines, patients present with various urological complications such as cysts and urinary tract infections, decreased glomerular filtration rate, chronic low back pain, and hypertension. ADPKD patients often present additional extrarenal conditions such as hepatic cysts (in over 90% of patients over 35 years old), pancreatic cysts, intracranial aneurysms, colonic diverticulosis, and cardiac valve defects. Overall, ADPKD is associated with significant morbidity and a shortened average lifespan.

[0004] ADPKD is primarily caused by mutations in either the polycystin gene PKD1 (polycystin 1, transient receptor potential channel interaction) (74–85% of patients) or PKD2 (15–26%). The phenotype of patients with PKD1 mutations is usually more severe than that of patients with PKD2 mutations, which is reflected in the approximately 20-year difference in the mean age of ESRD (54.3 years for PKD1 disease and 74.0 years for PKD2 disease).

[0005] There is an ongoing need for new treatments or preventive measures for ADPKD. [Overview of the project]

[0006] PKD1 consists of 46 exons spanning approximately 50 kb of genomic DNA on the short arm of chromosome 16 (16p13.3). The polycystin 1 protein encoded by standard PKD1 is a 4,303-amino acid glycosylated intrinsic membrane protein that localizes to primary cilia, endoplasmic reticulum, adherent and desmosome junctions, apical membrane, cell membrane, and binding complexes. Polycystin 1 contains a large N-terminal extracellular domain, multiple transmembrane domains, and a cytoplasmic C-terminal tail, and functions as a calcium-permeable cation channel and regulator of intracellular calcium homeostasis. It is also involved in the regulation of cell-cell / matrix interactions and G protein-coupled signaling pathways. Splice variants encoding different isoforms of this gene have attracted attention.

[0007] While we do not wish to be bound by theory, it is believed that renal cysts in ADPKD develop when the functional level of polycystin 1 falls below a certain level. In fact, this is consistent with the fact that the level of residual polycystin 1 function from the mutated gene directly correlates with the severity of the disease. The median age of onset for ESRD was 55 years for carriers of truncated mutations (complete loss of function) and 67 years for carriers of non-truncate mutations (partial loss of function).

[0008] This disclosure provides antisense oligonucleotides (ASOs), antisense RNA (AR) expression vectors, and related compositions and methods for increasing PKD1 mRNA and / or polycystin 1 protein levels by modulating the post-transcriptional or translational regulation of the 3' untranslated region (UTR) of PKD1 mRNA, for example, by blocking the specific binding of miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429) to their binding sites.

[0009] Accordingly, in one embodiment, provided herein is an ASO that binds to a target region of the 3' UTR of PKD1 mRNA, where the binding of the antisense oligonucleotide to the target region increases the level of polycystin 1 protein. In another embodiment, provided herein is an ASO that binds to a target region of the 3' untranslated region (UTR) of polycystic kidney disease 1 (PKD1) mRNA, where the binding of the antisense oligonucleotide to the target region reduces the growth or size of renal cysts when introduced into 3D renal cyst culture. In some examples, the binding of the antisense oligonucleotide to the target region reduces the specific binding of miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR. In some cases, if the binding of an antisense oligonucleotide to a target region reduces the specific binding of miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR, then the antisense oligonucleotide contains one of the sequences of SEQ ID NOs: 2-351 or 353-362.

[0010] In a further embodiment, provided herein is a vector for expressing an AR in mammalian cells that binds to a target region of the 3' UTR of PKD1 mRNA, where the binding of the AR to the target region increases the level of polycystin protein. In another embodiment, provided herein is a vector for expressing an AR in mammalian cells that binds to a target region of the 3' UTR of PKD1 mRNA, where the binding of the AR to the target region reduces cyst growth when introduced into 3D renal cyst culture. In some examples, the binding of the AR to the target region reduces the specific binding of miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR. In some examples, if the binding of the AR to the target region reduces the specific binding of miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR, the AR contains or consists of one of the sequences of SEQ ID NOs. 2-351 or 353-362. In some examples, the vector is a non-viral vector. In other examples, the vector is a viral vector. In some examples, if the vector is a viral vector, the viral vector is provided in a recombinant virus selected from the group consisting of adeno-associated viruses (AAVs), adenoviruses, lentiviruses, and aneroviruses.

[0011] In some cases, AR can be delivered by a vector (e.g., plasmid or recombinant virus) containing a renal cell type-selective or tissue-selective promoter to drive AR expression in mammalian cells. In some cases, the promoter is selective for expression in renal cells selected from the group consisting of pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending limb cells of the loop of Henle, descending limb cells of the loop of Henle, distal tubular cells, connecting tubular cells, interstitial cells, chief cells, peritubular capillary endothelial cells, and glomerular endothelial cells. In some cases, the vector contains an inducible promoter.

[0012] In some examples, in either the aforementioned ASO or vector, the ASO or AR binds to the target portion of the 3' UTR corresponding to SEQ ID NO: 1. In some examples, the nucleotide sequence of the ASO or AR is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the target portion over the length of the ASO or AR. In some examples, the nucleotide sequence of the ASO or AR contains or consists of one of SEQ ID NOs: 2-351 or 353-362. In some examples, the nucleotide sequence of the ASO or AR contains or consists of one of SEQ ID NOs: 17, 47, 73, 334-337, or 355-360. In some examples, the nucleotide sequence of the ASO or AR contains or consists of one of SEQ ID NOs: 334 or 335. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of sequence numbers 355-360. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of sequence numbers 17, 73, 336, and 337. In some cases, the nucleotide sequence of ASO or AR includes or consists of sequence number 47.

[0013] In some examples, any of the aforementioned ASOs include a skeletal modification. In some examples, the skeletal modification includes a phosphorothioate bond or a phosphorodiamidate bond. In other examples, the ASO includes a phosphorodiamidate morpholino, locked nucleic acid (LNA), peptide nucleic acid (PNA), or a 2'-O modification such as a 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some examples, the ASO includes at least one modified sugar moiety. In other examples, each sugar moiety within the ASO is a modified sugar moiety. In some examples, the ASO includes a 2'-O-methoxyethyl moiety. In other examples, each nucleotide in the ASO includes a 2'-O-methoxyethyl moiety.

[0014] In some cases, for either the aforementioned ASO or vector, the nucleotide sequence of the ASO or AR consists of 20-30 nucleotides, 22-30 nucleotides, 24-30 nucleotides, 25-30 nucleotides, or 26-30 nucleotides. In some cases, the nucleotide sequence of the ASO or AR consists of 25-30 nucleotides. In some cases, if the sequence of the ASO consists of 20-30 nucleotides, the ASO contains one or more phosphorodiamidate morpholino moieties.

[0015] In some examples, one of the aforementioned ASOs also includes a linked functional moiety. In some examples, the functional moiety includes a delivery moiety. In some examples, the delivery moiety is selected from the group consisting of lipids, peptides, carbohydrates, polyethers (e.g., polyethylene glycol), and antibodies. In some examples, if the ASO includes a delivery moiety, the delivery moiety includes a cell-permeable peptide (CPP). In some examples, the delivery moiety includes a receptor-binding domain (RBD). In some examples, the delivery moiety includes a poly(ethylene glycol) (PEG) moiety. In some examples, the delivery moiety includes an N-acetylgalactosamine (GalNAc) moiety. In some examples, the delivery moiety includes a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is approximately C8 to C20. In other examples, the functional moiety includes a stabilizing moiety. In some examples, the functional moiety is covalently bonded to the ASO. In other examples, the functional moiety is non-covalently bonded to the ASO. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO.

[0016] In a related embodiment, provided herein are pharmaceutical compositions comprising either the aforementioned ASO or vector and a pharmaceutically acceptable excipient.

[0017] In a further relevant embodiment, provided herein is a method for treating autosomal ADPKD, wherein a therapeutically effective amount of the aforementioned pharmaceutical composition is administered to a subject in need. In some examples, the subject to be treated is a human subject. In another relevant embodiment, provided herein is the use of either the aforementioned ASO or vector in the manufacture of a pharmaceutical product for treating ADPKD.

[0018] In another embodiment, provided herein is a method for increasing PKD1 mRNA and / or polycystin 1 protein in ex vivo cells or in vivo tissue, the method comprising contacting the cells or tissue with one of the aforementioned ASOs, vectors, or pharmaceutical compositions.

[0019] In yet another embodiment, provided herein are genetically modified cells comprising either the aforementioned ASO or vector. In some examples, the genetically modified cells are mammalian cells. In some examples, the genetically modified mammalian cells are genetically modified human cells. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 - Schematic diagram of the exon-intron structure of PKD1, miRNA binding sites in the PKD 3' UTR, and the relative binding sites of exemplary PMOs. (A) Exon / intron map of human PKD1 premRNA; (B) Sequence of PKD1 mRNA 3' UTR containing the binding sites of miR-17 family miRNAs (bold). Relative positions of complementary PMOs with sequences corresponding to SEQ ID NOs. 47 (+604+628), 334 (+611+635), and 335 (+615+639) are shown, respectively; (C) Sequence of PKD1 mRNA 3' UTR containing the binding sites of miR-200 family miRNAs (bold). The relative positions of complementary PMOs with sequences corresponding to sequence numbers 14 (+505+529), 337 (+515+539), 336 (+677+701), 73 (+682+706), and 75 (+688+712) are shown. [Figure 2]Figure 2 - Screening of PPMOs targeting the miR-17 binding site of PKD1 in HEK293 cells. Screening of PPMOs with sequences corresponding to SEQ ID NOs. 47 (+604+628), 334 (+611+635), and 335 (+615+639) in HEK293 cells. The graph shows PKD1 mRNA expression 24 hours after treatment with miR-17 site-targeted PPMO or non-targeted control (GTC CTR). PKD1 mRNA expression was normalized to TBP, and expression in untreated cells was set to 1. [Figure 3] Figure 3 - Effect of PPMO targeting a sequence within the 3' UTR of PKD1 to which the miR-17 miRNA seed sequence binds in ADPKD patient cell lines. PPMOs that showed significant PKD1 mRNA upregulation according to Example 2 were selected for their ability to upregulate polycystin 1 (PC1) protein in ADPKD patient cell lines with a PKD1 heterozygous mutation (p.Q2556*). The graph shows the median fluorescence intensity (MFI) of PC1 protein staining on the cell surface, measured by flow cytometry 5 days after treatment with 10 μM miR-17 site-targeted PPMO. The MFI of polycystin protein staining was normalized to an isotype-staining antibody control, and the MFI in untreated cells was set to 1. [Figure 4] Figure 4 - Functional validation of PPMO in a patient-derived 3D cyst model. Functional validation of peptide PMO (PPMO) with sequences corresponding to SEQ ID NOs. 47(+604+628), 334(+611+635), and 335(+615+639) in patient-derived cystic cells. PPMO was administered at 1 μM, 3 μM, 10 μM, and 20 μM concentrations. After 7 days of exposure, cultures were fixed and stained for actin cytoskeleton and nucleus. Cyst growth and swelling were visualized by high-content microscopy imaging. Representative images show cyst size in wells treated with 20 μM PPMO 7 days post-treatment. The assay was performed under non-stimulating conditions (spontaneous cyst formation). [Figure 5]Figure 5 - Quantification of patient-derived 3D cyst area and cell death. Images from all assay conditions described in Figure 4 were further analyzed to determine dose-dependent changes in cyst area (μm2) and cell death (%) to distinguish between efficacy and cytotoxicity. The cell death cutoff was set at 15%. (AC) Swelling inhibition was determined from the cyst area measurements. This was calculated as the average area of ​​each cyst in each plane of the z-stack. (DF) Cytotoxicity was calculated as the percentage of dead cells. Cells were scored as “dead” if the nucleus was not associated with co-localized actin cytoskeleton (rhodamine-phalloidin labeled). This value was normalized against the solvent control (0%) and presented as the mean + / - standard deviation of replication. [Figure 6] Figure 6. Screening of PPMO microwalks for inhibition of miR-17 binding. PPMOs corresponding to the oligo sequences of SEQ ID NOs. 353-361 were incubated with HEK293 cells for 24 hours at concentrations of 3 μM, 10 μM, and 30 μM (n=3 technical replicates per treatment condition). A non-targeted control PPMO designed not to hybridize to known human transcripts was used as a negative control treatment. This control PPMO was ligated to the same cell-permeable peptide as the test PPMO. A positive control oligonucleotide (RGLS4326, Med Chem Express, catalog no. HY-139290), an inhibitor of miR-17, was also included as an assay control. Data were normalized for two housekeeping genes and reported as relative levels compared to untreated cells set to 1. Data represent mean ± SD, UT = untreated cells. n=1 biological replicate. TBP = TATA-binding protein, DHX57 = DExH-Box helicase 57. [Figure 7]Figure 7: Screening of PPMOs for inhibition of miR-200 binding. PPMOs corresponding to the oligonucleotide sequences of SEQ ID NOs. 14, 73, 75, and 336-337 were incubated with HEK293 cells for 24 hours at concentrations of 3 μM, 10 μM, and 30 μM (n=3 technical replicates per treatment condition). A non-targeted control PPMO designed not to hybridize to known human transcripts was used as a negative control treatment. This control PPMO was ligated to the same cell-permeable peptide as the test PPMO. A positive control oligo (RGLS4326, Med Chem Express, catalog no. HY-139290), an inhibitor of miR-17, was also included. Data were normalized for two housekeeping genes and reported as relative levels compared to untreated cells set to 1. Data represent mean ± SD, UT = untreated cells. n=2 biological replicates. TBP = TATA-binding protein, DHX57 = DExH-Box helicase 57. [Figure 8] Figure 8: Western blot image of PPMO-treated HEK293 cells. HEK293 cells were treated with PPMO (+604+628) and (+611+635) containing sequences corresponding to sequence numbers 47 and 334, respectively. A non-targeted control (NTC) was used as a negative control, and a miR-17 inhibitor (RGLS4326, Med Chem Express, catalog no. HY-139290) was included as a positive control. PPMO-treated cells were harvested on day 5 and analyzed for PC1 expression using a Western blot assay. Two protein bands of approximately 462 kDa and 350 kDa represent the full-length (FL) and N-terminal fragment (NTF) of PC1, respectively. The intensity of total protein staining was analyzed as a loading control. The experiment was repeated four times (quadruplicate). [Figure 9]Figure 9: Quantification of PC1 protein upregulation in PPMO-treated HEK293 cells. The bar graph represents the mean ± S.D. of PC1 protein expression analyzed using the gel images of Figure 8. Both FL and NTF bands were included in the analysis and normalized against total protein staining. The dashed line indicates the untreated baseline PC1 protein level set as 1. Experiments were performed in quadruplicate. n = 2 biological replicates. UT = untreated cells. NTC = non-target control. [Figure 10] Figure 10: PC1 protein analysis in PPMO-treated WT9-7 cells. WT9-7 cells were incubated with PPMO (4 replicates) corresponding to the sequence of SEQ ID NO: 47 (+604+628) and non-target control (NTC) for 2, 3, or 5 days. Non-target control (NTC) was used as a negative control and an inhibitor of miR-17 (RGLS4326, Med Chem Express, catalog number HY-139290) was included as a positive control. The bar graph represents the mean + S.D. of PC1 protein normalized against total protein staining compared to untreated cells. n = 1 biological replicate. UT = untreated cells. NTC = non-target control.

Mode for Carrying Out the Invention

[0021] Detailed Description General Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition of matter, series of steps, or series of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, compositions of matter, series of steps, or series of compositions of matter. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes one and two or more, reference to "an" includes one and two or more, reference to "the" includes one and two or more, and so forth.

[0022] Each example described herein shall apply mutatis mutandis to all other examples unless otherwise specified.

[0023] Those skilled in the art will understand that the disclosure herein is subject to changes and modifications other than those specifically described. It should be understood that this disclosure includes all such changes and modifications. This disclosure also includes all processes, features, compositions, and compounds mentioned or indicated herein, individually or collectively, as well as any combination of any two or more of the aforementioned processes or features.

[0024] This disclosure is not limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods as described herein are clearly within the scope of this disclosure. This disclosure is carried out without excessive experimentation using conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid-phase peptide synthesis, and immunology, unless otherwise indicated. Such techniques are described and explained throughout the literature of sources such as Perbal 1984, Sambrook et al., 2001, Brown (ed.) 1991, Glover and Hames (eds.) 1995 and 1996, Ausubel et al. (including all updates to date), Coligan et al. (ed.) (including all updates to date), Maniatis et al. 1982, Gait (ed.) 1984, Hames and Higgins (eds.) 1984, and Freshney (ed.) 1986.

[0025] The term "and / or," for example "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and shall be deemed to explicitly support both meanings or either of them.

[0026] The term "approximately" refers to + / - 20%, more preferably + / - 10%, of the specified value, unless otherwise specified. To avoid ambiguity, if a specified value follows "approximately," it is interpreted as including that specified value exactly (for example, "approximately 10" includes exactly 10).

[0027] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” is understood to mean that it includes the elements, integers, or processes, or groups of elements, integers, or processes described, but not that it excludes other elements, integers, or processes, or groups of elements, integers, or processes.

[0028] As used herein, the terms “antisense oligonucleotide,” “antisense oligomer,” or “ASO” encompass oligonucleotides and other oligomer molecules containing nucleic acid bases that can hybridize to a complementary sequence on a target RNA transcript, including but not limited to those that do not contain a sugar moiety, such as peptide nucleic acids (PNAs). Preferably, the ASO is an ASO that is resistant to nuclease cleavage or degradation.

[0029] As used herein in relation to ASO or AR, the terms “binding to target region” or “binding within target region” refer to specific hybridization between an ASO or AR nucleotide sequence and a complementary target nucleotide sequence within the scope described herein. In some cases, specific hybridization occurs under ex vivo conditions when the hybridization occurs under high stringency conditions. “High stringency conditions” means that under such ex vivo conditions, the ASO or AR hybridizes to the target sequence more strongly than nonspecific hybridization. Thus, high stringency conditions are those that distinguish polynucleotides with precisely complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences where a few small regions (e.g., 1-5 bases) coincidentally match the probe. Such small complementary regions melt more readily than full-length complements of 12-17 bases or more and are readily distinguishable by hybridization of moderate stringency. For example, high stringency conditions include low-salt and / or high-temperature conditions provided by, for instance, about 0.02–0.1 M NaCl or equivalent, and temperatures of about 50–70°C. Those skilled in the art will understand that under in vivo conditions, the specificity of hybridization between an ASO or AR and its target sequence is defined in terms of the level of complementarity between the ASO or AR and the target sequence with which it hybridizes within the cell.

[0030] The term "peptide" is intended to include compounds composed of amino acid residues linked by amide bonds. Peptides may be natural or unnatural, ribosomal coded or synthetic. Typically, peptides consist of 2 to 200 amino acids. For example, a peptide may have a length in the range of 10 to 20 amino acids, or 10 to 30 amino acids, or 10 to 40 amino acids, or 10 to 50 amino acids, or 10 to 60 amino acids, or 10 to 70 amino acids, or 10 to 80 amino acids, or 10 to 90 amino acids, or 10 to 100 amino acids, including any length within the said range. A peptide may contain, or consist of, less than about 150 amino acids, or less than about 125 amino acids, or less than about 100 amino acids, or less than about 90 amino acids, or less than about 80 amino acids, or less than about 70 amino acids, or less than about 60 amino acids, or less than about 50 amino acids.

[0031] The peptides referred to herein include “inverso” peptides in which all L-amino acids are replaced with corresponding D-amino acids, and “retroinverso” peptides in which the amino acid sequence is reversed and all L-amino acids are replaced with D-amino acids.

[0032] Peptides may contain both L- and / or D-type amino acids. For example, both L- and D-type amino acids may be used for different amino acids within the same peptide sequence. In some examples, the amino acids in a peptide sequence are L-type, like natural amino acids. In some examples, the amino acids in a peptide sequence are a combination of L- and D-type. Furthermore, peptides may contain rare but naturally occurring amino acids, including but not limited to hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, and pyroglutamic acid (Pyr). Peptides may also incorporate non-natural amino acids, including but not limited to homoamino acids, N-methylamino acids, alpha-methylamino acids, beta (homo)amino acids, gammaamino acids, and N-substituted glycines. Peptides may be linear or cyclic peptides.

[0033] The term "protein" includes a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to one another by covalent or non-covalent bonds (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using appropriate chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0034] The percentage of amino acid sequence identity for a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum possible sequence identity percentage, and without considering conservative substitutions as part of the sequence identity. Amino acid sequence identity can be determined using the EMBOSS pairwise alignment algorithm tool, available from the European Bioinformatics Laboratory (EMBL-EBI), part of the European Molecular Biology Laboratory. This tool is accessible at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings, including Gap Open: 10.0 and Gap Extend 0.5, are used. The default matrix "Blosum62" is used for the amino acid sequence.

[0035] The term "cell-permeable peptide" (CPP) refers to peptides that can pass through cell membranes. In one example, a CPP can travel across the mammalian cell membrane and enter the cell. In another example, a CPP can direct its conjugate to a desired intracellular compartment. Thus, CPPs can direct or facilitate the passage of target molecules across phospholipid membranes, mitochondrial membranes, endosomal membranes, lysosomal membranes, vesicle membranes, or nuclear membranes. CPPs can travel across membranes with their amino acid sequence intact or partially degraded.

[0036] CPPs can deliver target molecules, such as ASOs disclosed herein, from the extracellular space through the plasma membrane to the cytoplasm or a desired intracellular compartment. Alternatively, or in addition, CPPs can deliver target molecules across epithelial, endothelial, basement membrane, transmucosal, cardiovascular, cutaneous, gastrointestinal, and / or pulmonary barriers. In some embodiments, CPPs are selectively targeted to or taken up by the kidney.

[0037] The terms "peptide ligand" or "receptor-binding domain" refer to peptides that can bind to membrane surface receptors and enable the movement of peptides across the cell membrane. In one example, a peptide ligand may enable movement across the cell membrane via the innate endocytosis of the target receptor. In another example, a peptide ligand may utilize complementary mechanisms of movement across the cell membrane, including the use of bound CPPs. In one example, a peptide ligand has the ability to move across mammalian cell membranes and enter cells. In yet another example, a peptide ligand may direct its conjugate to a desired intracellular compartment. Thus, peptide ligands can direct or facilitate the cellular uptake of target molecules across phospholipid membranes, mitochondrial membranes, endosomal membranes, lysosomal membranes, vesicle membranes, or nuclear membranes. Peptide ligands can move across membranes with their amino acid sequence intact or partially degraded.

[0038] Peptide ligands can, via binding to target receptors, guide target molecules such as ASOs disclosed herein from the extracellular space through the plasma membrane into the cytoplasm or a desired intracellular compartment. Alternatively, or in addition, peptide ligands can, via binding to target receptors, guide target molecules across relevant biological barriers, such as the renal basement membrane, blood-brain barrier, transmucosal, blood-retinal, cardiovascular, skin, gastrointestinal, and / or pulmonary barriers.

[0039] Composition for increasing PKD1 mRNA and polycystin-1 protein levels MicroRNAs (miRNAs) are a family of short (19-23 nucleotides) non-coding single-strand unstable RNAs. While they can bind to any portion of a target mRNA, their primary mechanism of action is to regulate gene expression by binding to a complementary RNA sequence in the 3' untranslated region (3' UTR) and stimulating either mRNA degradation or translational repression. Both mechanisms lead to decreased expression of the target gene. In the case of the polycystin 1 (PKD1) gene encoding the polycystin 1 protein, miRNAs that bind to the 3' UTR of the encoding mRNA transcript include the miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) and the miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429).

[0040] While we do not wish to be bound by theory, it is thought that an antisense sequence at least partially complementary to the microRNA binding site located within the PKD1 mRNA 3' UTR hybridizes to the PKD1 3' UTR, sterically inhibiting ("masking") the access of these miRNAs to the binding site on the PKD1 3' UTR, ultimately enabling an increase in polycystin 1 protein levels. Disclosed herein are ASOs that bind to a target region of the PKD1 mRNA 3' UTR, where the binding of an antisense oligonucleotide to the target region increases the levels of PKD1 mRNA and / or polycystin 1 protein. In some preferred examples, the ASO hybridizes to the target region of the PKD1 mRNA 3' UTR, thereby preventing one or more miRNAs from hybridizing to their specific target sequence, resulting in an increase in the levels of PKD1 mRNA and polycystin 1 protein.

[0041] Also disclosed herein is a vector for the expression of AR in mammalian cells, which binds to the target region of the 3' UTR of PKD1 mRNA, ultimately resulting in an increase in polycystin 1 protein levels as described above.

[0042] For reference, the sequence of a standard human PKD1 mRNA transcript ("PKD-201") is publicly available through the online Ensembl database under record ENST00000262304.9. The nucleotide sequence of the standard human PKD1 mRNA 3' UTR sequence is provided herein as Sequence ID No. 1.

[0043] Antisense oligonucleotides (ASOs) and antisense RNAs (ARs) In some preferred examples of the compositions and methods described herein, the ASO and AR have sequences that are fully complementary to the target sequence over their length, or nearly complementary (e.g., sufficiently complementary to bind to the target sequence and interfere with miRNA binding at the PKD1 mRNA 3' UTR binding site). The ASO and AR are designed to bind (hybridize) to the target RNA sequence (e.g., the target portion of a premRNA transcript) and remain hybridized under physiological conditions. Appropriate sequence selection of the ASO and AR generally avoids, where possible, similar nucleic acid sequences in other (i.e., off-target) locations in the genome or in cellular mRNA or miRNA, so as to limit the possibility of the ASO or AR hybridizing to such sites.

[0044] In some cases, the ASO or AR is "specifically hybridizes" or "specific" to the target portion of the target nucleic acid or PKD1 mRNA 3' UTR. Given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to complementary oligonucleotides.

[0045] ASO and AR sequences are “complementary” to their target sequences if hybridization occurs in antiparallel configuration between two single-stranded polynucleotides of complementary sequences. Complementarity can be quantified as the percentage (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules. The nucleotide sequences of ASO or AR do not need to be 100% complementary to their target nucleic acid in order to hybridize. In certain examples, the nucleotide sequences of ASO or AR in the compositions disclosed herein may have sequence complementarity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% over the length of the ASO or AR nucleotide sequence to the nucleotide sequence of the target portion of the RNA transcript. For example, an ASO or AR sequence in which 18 of the 20 nucleotides are complementary to the target region and therefore specifically hybridizes represents 90 percent complementarity. In such an example, the remaining non-complementary nucleotides of the ASO or AR may be clustered together or scattered with the complementary nucleotides, and do not need to be contiguous. The complementarity of an ASO or AR sequence to a target nucleotide sequence (expressed as a "percentage of complementarity" to that target sequence, or a "percentage of identity" to its inverse complementary sequence) can be routinely determined using algorithms known in the art, as exemplified by the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., 1990, J. Mol. Biol., 215:403-410; Zhang et al., 1997, Genome Res., 7:649-656).

[0046] In some examples, the ASO or AR does not hybridize to all nucleotides in the target sequence, and the nucleotide positions to which it hybridizes may be continuous or discontinuous. The ASO or AR may hybridize across one or more segments of the mRNA 3' UTR region such that intervening or adjacent segments do not participate in the hybridization event (e.g., a loop or hairpin structure may be formed). In some examples, the nucleotide sequences of the ASO or AR described herein are complementary to the target region of the PKD1 mRNA 3' UTR. In some preferred examples, the ASO or AR is complementary to the target region of the PKD1 mRNA 3' UTR corresponding to SEQ ID NO: 1. In some examples, the nucleotide sequences of the ASO or AR are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the target region of the PKD1 3' UTR over the length of the ASO or AR. In some cases, the nucleotide sequences of ASO or AR have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over the full length of either sequence provided by SEQ ID NOs 2-351 or 353-362 (shown in Table 1).

[0047] The ASO or AR for use in the compositions described herein may be of any length suitable for specific hybridization to the target sequence. In some examples, the nucleotide sequence of the ASO or AR consists of 8 to 50 nucleotides. For example, the ASO or AR sequence may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleotides in length. In some examples, the ASO is longer than 50 nucleotides but no more than 100 nucleotides.In some examples, the ASO or AR nucleotide sequence is 8-50 nucleotides, 8-40 nucleotides, 8-35 nucleotides, 8-30 nucleotides, 8-25 nucleotides, 8-20 nucleotides, 8-15 nucleotides, 9-50 nucleotides, 9-40 nucleotides, 9-35 nucleotides, 9-30 nucleotides, 9-25 nucleotides, 9-20 nucleotides, 9-15 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-35 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, 10-15 nucleotides, 11-50 nucleotides, 11-40 nucleotides, 11-35 nucleotides, 11-30 nucleotides, 11-25 nucleotides, 11-20 nucleotides, 11-15 nucleotides, 12-50 nucleotides, 12-40 nucleotides, 12-35 nucleotides. Chidos are 12-30 nucleotides, 12-25 nucleotides, 12-20 nucleotides, 12-15 nucleotides, 13-50 nucleotides, 13-40 nucleotides, 13-35 nucleotides, 13-30 nucleotides, 13-25 nucleotides, 13-20 nucleotides, 14-50 nucleotides, 14-40 nucleotides, 14-35 nucleotides, 14-30 nucleotides, 14-25 nucleotides, 14-20 nucleotides, 15-50 nucleotides, 15-40 nucleotides, 15-35 nucleotides, 15-30 nucleotides, 15-25 nucleotides, 15-20 nucleotides, 20-50 nucleotides, 20-40 nucleotides, 20-35 nucleotides, 20-30 nucleotides, 20-25 nucleotides, 25-50 nucleotides, 25-40 nucleotides, 25-35 nucleotides, or 25-30 nucleotides in length. In some examples, ASO or AR is 20 nucleotides in length. In some preferred examples, the nucleotide sequence of ASO or AR is 25 nucleotides long.

[0048] In some examples, for each occurrence of "G" in the ASO or AR sequences disclosed herein, "G" is guanosine or inosine. In some examples, for each occurrence of "T" in the ASO or AR sequences disclosed herein, "T" is one of thymidine, inosine, uracil, or an isomer or modified form of uracil (e.g., pseudouridine or N1-methylpseudridine). In some examples, for each occurrence of "C" in the ASO or AR sequences disclosed herein, "C" is cytosine or a modified form of cytosine (e.g., 5'-methylcytosine).

[0049] In some cases, the nucleotide sequence of ASO or AR includes one of the sequences of SEQ ID NOs: 2-351 or 353-362. In some cases, the nucleotide sequence of ASO or AR consists of one of the sequences of SEQ ID NOs: 2-351 or 353-362. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of the sequences of SEQ ID NOs: 17, 47, 73, 334-337, or 355-360. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of the sequences of SEQ ID NOs: 334 or 335. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of the sequences of SEQ ID NOs: 355-360. In some cases, the nucleotide sequence of ASO or AR includes or consists of one of the sequences of SEQ ID NOs: 17, 73, 336, and 337. In some cases, the nucleotide sequence of ASO or AR includes or consists of SEQ ID NOs: 47.

[0050] ASO chemistry and modification The ASOs used in the compositions described herein may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination thereof. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides having modified or substituted sugar groups, and / or nucleotides having modified skeletons. In some examples, all nucleotides in the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the compositions and methods described herein are known in the art, for example, disclosed in U.S. Patent No. 8,258,109, U.S. Patent No. 5,656,612, U.S. Patent Application Publication No. 2012 / 0190728, and Roberts et al., 2020, Nature Rev. Drug Disc., 19:673-694.

[0051] One or more nucleotides of the ASO may be any naturally occurring unmodified nucleic acid base, such as adenine, guanine, cytosine, thymine, uracil, and inosine, or any synthetic or modified nucleic acid base that is sufficiently similar to an unmodified nucleic acid base to form hydrogen bonds with a nucleic acid base present on the target RNA transcript. Suitable examples of modified nucleic acid bases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0052] ASOs contain a “skeleton” structure that refers to the bond between the nucleotide / monomer of the ASO. In naturally occurring oligonucleotides, the skeleton contains a 3'-5' phosphodiester bond that links the sugar moieties of adjacent nucleotides. Suitable types of skeleton bonds for ASOs described herein include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, phosphorodiamidates, phosphoroselenoates, phosphorodyselenoates, phosphoranilothiolates, phosphoraniladates, and phosphoramidates. In some examples, the skeleton modification is a phosphorothioate bond. In other examples, the skeleton modification is a phosphorodiamidate bond. See, for example, Roberts et al., op. cit., and Agrawal (2021), Biomedicines, 9:503. In some examples, the skeleton structure of ASOs does not contain a phosphorus-based bond, but rather contains a peptide bond, as in peptide nucleic acids (PNAs), or linking groups containing carbamates, amides, and linear and cyclic hydrocarbon groups.

[0053] In some examples, the stereochemistry of the inter-phosphorus nucleotide bonds in the ASO skeleton is random. In other examples, the stereochemistry of the inter-phosphorus nucleotide bonds in the ASO skeleton is controlled and not random. For example, U.S. Patent No. 9,605,019 describes a method for independently selecting the chirality handedness of each phosphorus atom in an oligonucleotide. In some examples, the ASOs used in the compositions and methods provided herein, including but not limited to the ASO sequence disclosed as SEQ ID NO: 2-351, are ASOs having non-random phosphodiester nucleotide bonds. In some examples, the compositions used in the compositions or methods disclosed herein include pure diastereomer ASOs. In other examples, the composition comprises an ASO having a diastereomer purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0054] In some examples, ASOs have a non-random mixture of Rp and Sp configurations in their phosphonucleotide interbonding. In some examples, ASOs used in compositions and methods disclosed herein contain about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp, or about 100% Rp.

[0055] In some examples, the ASOs described herein include a sugar moiety containing ribose or deoxyribose, or a modified sugar moiety or sugar analog containing a morpholine ring. Suitable examples of modified sugar moieties include, but are not limited to, 2'-O-modifications, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidates, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and 2'-substitutions such as bicyclic-modified sugars. In some examples, the sugar moiety is selected from 2'-O-Me, 2'F, and 2'MOE. In other examples, the sugar moiety is an additional crosslinking, such as locked nucleic acid (LNA). In some examples, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some examples, the sugar moiety contains ribofuranosyl or 2'-deoxyribofuranosyl modification. In some examples, the sugar moiety contains 2'-4'-restricted 2'-O-methyloxyethyl (cMOE) modification. In some examples, the sugar moiety contains cEt 2',4'-restricted 2'-O-ethyl BNA modification. In other examples, the sugar moiety contains tricycloDNA (tcDNA) modification. In some examples, the sugar moiety contains ethylene nucleic acid (ENA) modification. In some examples, the sugar moiety contains 2'-O-(2-N-methylcarbamoylethyl) (MCE). The modifications are known in the art, as exemplified in Jarver et al., 2014, Nucleic Acid Therapeutics, 24(1): 3747.

[0056] In some examples, each constituent nucleotide of the ASO is modified in the same way, for example, all bonds in the ASO skeleton contain phosphorothioate bonds, or each ribose sugar moiety contains a 2'-O-methyl modification. In other examples, different combinations of modifications are used, for example, an ASO containing a combination of a phosphorodiamidate bond and a sugar moiety (morpholino) containing a morpholine ring. In some examples, the ASO contains one or more skeleton modifications. In some examples, the ASO contains one or more subsugar modifications. In some examples, the ASO contains one or more skeleton modifications and one or more subsugar modifications. In some examples, the ASO contains a 2'-O-MOE modification and a phosphorothioate skeleton. In some examples, the ASO contains a peptide nucleic acid (PNA).

[0057] In some preferred examples, ASOs include phosphorodiamidate morpholino (PMO). Those skilled in the art will understand that ASOs can be modified to achieve desired properties or activities, or to reduce undesirable properties or activities. In some examples, ASOs are modified to alter one or more properties. For example, such modifications can enhance binding affinity to target sequences on premRNA transcripts; reduce binding to any non-target sequences; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of ASOs into cells and / or specific intracellular compartments; alter the pharmacokinetics or pharmacodynamics of ASOs; and / or regulate the half-life of ASOs in vivo.

[0058] In some examples, the ASO comprises one or more 2'-O-(2-methoxyethyl)(MOE) phosphorothioate-modified nucleotides, which have been shown to confer a significant improvement in the ASO's resistance to nuclease degradation and increased bioavailability. Methods for the synthesis and chemical modification of ASOs, as well as for the synthesis of ASO conjugates, are well known in the art, and such ASOs are commercially available. In some examples, the compositions provided herein (e.g., pharmaceutical compositions) comprise two or more ASOs having different chemical properties but complementary to the same target portion of the PKD1 mRNA 3' UTR. In other examples, the compositions comprise two or more ASOs complementary to different target portions of the PKD1 mRNA 3' UTR.

[0059] In some examples, the compositions disclosed herein include ASOs linked to a functional portion. In some examples, the functional portion is a delivery portion, a targeting portion, a detection portion, a stabilizing portion, or a therapeutic portion. In some examples, the functional portion includes a delivery portion or a targeting portion. In some examples, the functional portion includes a stabilizing portion. In some preferred examples, the functional portion is a delivery portion. Suitable delivery portions include, but are not limited to, lipids, peptides, carbohydrates, polyethers, and antibodies.

[0060] In some cases, the delivery portion includes a cell-permeable peptide (CPP). A suitable example of a CPP is described, for example, in PCT / AU2020 / 051397. In some cases, the amino acid sequence of the CPP includes or consists of RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 352). In one example, the CPP includes the sequence RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 352), where any amino acid other than glycine is optionally a D amino acid. In other examples, the delivery portion includes a receptor-binding domain.

[0061] In other examples, the delivery portion includes a carbohydrate. In some examples, the carbohydrate delivery portion is selected from N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), and mannose. In one example, the carbohydrate delivery portion is GalNAc. In other examples, the delivery portion includes a lipid. Suitable lipid examples as the delivery portion include, but are not limited to, cholesterol moieties, cholesteryl moieties, and aliphatic lipids. In some examples, the delivery portion includes a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is approximately C8–C20. Examples of suitable fatty acid moieties and their binding to oligonucleotides can be found, for example, in International Patent Publication WO 2019232255 and Prakash et al., (2019).

[0062] In further examples, the delivery portion includes an antibody, as described, for example, in Dugal-Tessier et al. (2021). Suitable examples of stabilizing portions include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and poly(2-oxazoline) (POx). In some examples, when the ASO is linked to the functional portion, the functional portion is covalently bonded to the ASO. In other examples, the functional portion is acovalently bonded to the ASO.

[0063] The functional moiety can be linked to one or more nucleotides in the ASO at any of several positions on a sugar, base, or phosphate group, for example, using a linker, as understood and described in the literature in the art. The linker may include a divalent or trivalent branched linker. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO. In some examples, a composition comprising any of the ASOs disclosed herein also includes a delivery nanocarrier complexed with the ASO. In some examples, the delivery nanocarrier is selected from lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In other examples, the delivery nanocarrier includes lipid nanoparticles that encapsulate the ASO. Various delivery ASO-nanocaracher complex formats are known in the art, for example, Roberts et al., as outlined above.

[0064] PKD1 antisense RNA (AR) expression vector In some examples, provided herein are compositions comprising a vector for expressing an AR that binds to a target region of the PKD1 3' UTR in mammalian cells, where the binding of the AR to the target region reduces the specific binding of miRNAs to the 3' UTR, such as miR-17 family miRNAs (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or miR-200 family miRNAs (e.g., miR-200b, miR200c, or miR-429). In some examples, the promoter used in the expression vector is a renal cell type selective promoter for driving AR expression in mammalian cells. In some cases, renal cell type-selective promoters are selective for expression in renal cell types selected from a list consisting of pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending limb cells of the loop of Henle, descending limb cells of the loop of Henle, distal tubular cells, connecting tubular cells, interstitial cells, chief cells, peritubular capillary endothelial cells, and glomerular endothelial cells. Examples include the sodium-dependent phosphate transporter type 2a (NPT2a) promoter for expression in the proximal tubule, the sodium-potassium-2-chloride cotransporter (NKCC2) promoter for expression in the thick ascending limb of the loop of Henle (TALH), the aquaporin 2 (AQP2) promoter for expression in the collecting duct, and the podosin promoter for expression in podocytes.

[0065] In some cases, the promoter can be inducible by an inducible promoter, such as a ligand-controlled transactivator like the Tet-inducible rtTA, which enables titration of AR transcription in target mammalian cells. In some cases, the promoter driving AR expression is the U6 or other Pol III promoter, which is particularly suitable for the transcription of short RNA sequences such as the AR sequences disclosed herein. In some cases, the expression vector utilizes a hybrid promoter system, such as the Tet-O controlled U6 promoter system described in Lin et al. (2004), FEBS Letters, 577 (2004) 376-380. In some cases, when both cell type specificity and inducibility of the AR expression vector are desired, a two-part expression system is used in which the expression of a ligand-controlled transactivator is driven by a cell type-selective promoter, and the expression of the AR disclosed herein is driven by a promoter regulated by the ligand-controlled transactivator.

[0066] In some examples, the expression vectors used in the compositions disclosed herein are non-viral expression vectors, such as plasmid vectors, minicircle DNA vectors, and linear amplicon expression cassettes. In some examples, compositions containing non-viral expression viruses further include transfection agents. Exemplary transfection agents for transfection include, but are not limited to, jet-PEI® (available from Polyplus-transfection® SA, Strasbourg, France); TurboFect in vivo Transfection Reagent (ThermoFisher); and cationic derivatives of polyisoprenoid alcohols (PTAIs), such as those described in Rak et al. (2016), J Gene Med, 18(11-12):331-342.

[0067] In other examples, the expression vector used is a viral vector, i.e., a non-replicating recombinant virus suitable for the expression of the AR disclosed herein. Preferably, the recombinant virus for PKD1 AR expression is a DNA virus. Suitable types of DNA viruses include adeno-associated viruses (AAVs), adenoviruses, lentiviruses, herpes simplex viruses (HSVs), and aneroviruses. Methods for designing, producing, and using such types of recombinant DNA viruses are established in the art, as exemplified by Fukazawa et al. (2010), International J of Mol. Med, 25(1), 3-10 and “Gene Therapy Protocols” for adenoviruses; “Adeno-Associated Virus: Methods and Protocols” for AAV; Cody et al. (2013), Journal of Genetic Syndromes & Gene Therapy, 4(1), 126 and “Herpes Simplex Virus: Methods and Protocols” for HSV; and “Gene Therapy Protocols Vol. 1: Production and In Vivo Applications of Gene Transfer Vectors,” Merten et al. (2016), Molecular Therapy - Methods & Clinical Development, 3, 16017, and Emeagi et al. (2013), Current Molecular Medicine 13(4), 602-625 for lentiviruses. In some preferred examples, the viral vector is recombinant AAV.

[0068] Genetically modified cells Genetically modified cells are also provided herein. In some examples, genetically modified cells are genetically modified bacterial cells (e.g., recombinant Escherichia coli for amplifying the AR expression vectors disclosed herein). In other examples, genetically modified cells are genetically modified mammalian cells transfected with either the ASO or non-viral AR expression vectors disclosed herein, or transduced with either the viral AR expression vectors. In some examples, genetically modified mammalian cells exist ex vivo, for example, as a population of cultured cells. In other examples, genetically modified mammalian cells exist in vivo, for example, in mice. In some examples, genetically modified mammalian cells are human cells.

[0069] In some cases, genetically modified mammalian cells are primary cell types. Suitable examples of primary cell types include, but are not limited to, cystic cells, pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending limb cells of the loop of Henle, descending limb cells of the loop of Henle, distal tubular cells, connecting tubular cells, interstitial cells, chief cells, peritubular capillary endothelial cells, and glomerular endothelial cells. In some cases, such primary cell types can be obtained by differentiation of human pluripotent stem cell lines, such as human induced pluripotent stem cell (hiPSC) lines or human embryonic stem cell (hESC) lines. Methods for obtaining a variety of different renal cell types are known in the art, as outlined, for example, by de Carvalho Ribeiro et al. (2020) and Osafune et al. (2021). In some cases, primary cell types originate from dividing renal tissue, such as renal cysts. Cystic cells excised from ADPKD kidneys have been widely used since the 1980s. A detailed protocol for in vitro cyst formation was recently published by Sharma et al. (2019). In other examples, genetically modified mammalian cells are derived from cell lines. In some examples, the cell lines are pluripotent stem cell lines (e.g., hiPSC or hESC) or human kidney cell lines. In some examples, genetically modified mammalian cells are derived from HEK293, HK-2, or WT9-7 cell lines. In some preferred examples, genetically modified mammalian cells endogenously express polycystin 1. In some embodiments, genetically modified human cells are provided in kidney organoids, such as kidney organoids induced by differentiation of human pluripotent or human adult stem cells, as outlined, for example, in Kang (2023), Development & Reproduction, 27(2):57-65.

[0070] The genetically modified cells disclosed herein can be genetically modified by any of the many methods and strategies known in the art, such as transient transfection, stable transfection, and viral transduction. In some examples, transfection with ASO or nonviral vectors is carried out by nucleofection. In other examples, cell transfection is carried out by lipofection.

[0071] Pharmaceutical composition This specification also provides pharmaceutical compositions formulated with any of the aforementioned ASOs, nonviral expression vectors, modified messenger RNA (mmRNA), and viral expression vectors, together with at least pharmaceutically acceptable excipients, including carriers, fillers, preservatives, adjuvants, solubilizers, and / or diluents.

[0072] Pharmaceutical compositions comprising any of the ASOs or expression vector compositions described herein for use in the manner disclosed herein can be prepared according to the prior art known in the pharmaceutical industry and described in the published literature. In some examples, a pharmaceutical composition for treating a subject contains a therapeutically effective amount of any ASO or expression vector disclosed herein. Pharmaceutically acceptable salts are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and lac. This includes salts such as tobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium.Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfons, and aryl sulfons, where appropriate.

[0073] In some examples, the pharmaceutical composition is formulated into one of a number of possible dosage forms, including but not limited to topical ointments, intravenous solutions, subcutaneous injections, intrathecal administrations, intracisional administrations, tablets, capsules, gel capsules, liquid syrups, and softgels. In some examples, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. The aqueous suspension may further contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer. In some examples, the pharmaceutical formulations disclosed herein are provided in forms including but not limited to solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0074] In some examples, a pharmaceutical formulation comprising either an ASO or an expression vector as described herein may contain one or more penetration enhancers, carriers, excipients, or other suitable active or inactive components known to those skilled in the art. In some examples, where the pharmaceutical composition comprises liposomes, such liposomes may also include sterically stabilized liposomes, such as liposomes containing one or more specialty lipids. These specialty lipids result in liposomes with enhanced circulating lifespan. In some examples, the sterically stabilized liposomes may contain one or more glycolipids or be derivatized with one or more hydrophilic polymers, such as PEG moieties. In some examples, the pharmaceutical formulation may contain surfactants.

[0075] In some cases, the pharmaceutical composition also includes penetration enhancers to enhance the delivery of ASOs or nonviral expression vectors, for example, to aid diffusion across cell membranes and / or to increase the permeability of lipophilic drugs. In some cases, penetration enhancers include surfactants, fatty acids, bile salts, or chelating agents. In some examples, the pharmaceutical composition contains an ASO or nonviral vector in a dose ranging from about 0.0001 mg / kg to about 80 mg / kg, for example, 0.005 mg / kg, 0.007 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 77 mg / kg, or another dose in the range of about 0.01 mg / kg to about 80 mg / kg.

[0076] In some examples, the pharmaceutical composition includes multiple ASO or AR expression vectors. In some examples, in addition to the ASO or AR expression vectors, the pharmaceutical composition includes another drug or therapeutic agent suitable for treating a subject suffering from an inflammation-related condition.

[0077] method As described herein, ADPKD is associated with a deficiency in the level of functional polycystin 1. Therefore, the methods described herein include methods for treating ADPKD by administering a therapeutically effective amount of a pharmaceutical composition containing either the ASO or expression vector disclosed herein to the target. Similarly, in several examples, either the ASO or AR expression vector disclosed herein is used in the manufacture of pharmaceuticals for reducing inflammation.

[0078] Also provided herein are methods for increasing the levels of PKD1 mRNA and subsequently polycystin 1 protein in ex vivo cells or in vivo tissues, the methods comprising contacting cells with an ASO, AR expression vector, or pharmaceutical composition disclosed herein, thereby reducing cellular abnormalities associated with ADPKD, such as cyst formation. In some examples, administration to a subject or contact with cells increases the level of PKD1 mRNA or polycystin 1 protein by about 1.1 to about 3 times, for example, 1.3 to about 2.7 times, about 1.4 to about 2.6 times, about 1.5 to about 2.5 times, about 1.6 to about 2.4 times, about 1.7 to about 2.3 times, about 1.8 to about 2.2 times, or another increase of about 1.1 to about 3 times, compared to the level in tissue without administration or contact, by any of the ASO, AR expression vector, or pharmaceutical composition disclosed herein.

[0079] Appropriate routes of administration for treatment with the compositions, pharmaceutical compositions, or medicinal products disclosed herein include, but are not limited to, intravenous, intra-arterial, subcutaneous, intrathecal, oral, and topical. In some examples, any of the therapeutic methods disclosed herein may optionally include a step of determining the levels of PKD1 mRNA, polycystin 1 protein in a subject before and / or after treatment. As those skilled in the art will understand, the therapeutic methods disclosed herein include administering the compositions and pharmaceutical compositions disclosed herein to a subject (e.g., a human subject) in a therapeutically effective dose. The terms “effective dose” or “therapeutically effective dose,” as used herein, refer to a sufficient amount of the disclosed ASO, non-viral, or viral expression vector administered to alleviate to some extent one or more symptoms and / or clinical indicators associated with pathological inflammation in a particular disease or health condition. In some examples, “effective dose” for therapeutic use is one amount of the aforementioned agent necessary to provide a clinically significant reduction in disease symptoms and / or inflammatory markers, or to prevent disease symptoms, without excessive adverse side effects. The appropriate “effective dose” in individual cases may be determined using techniques such as dose escalation studies. The term “therapeutic effective dose” includes, for example, a prophylactic effective dose. It is understood that the “effective dose” or “therapeutic effective dose” may vary from subject to subject due to variations in the metabolism of any of the compounds, as determined by the prescribing physician, age, weight, the subject’s general condition, the condition being treated, the severity of the condition being treated, and the prescribing physician. For example only, the therapeutic effective dose may be determined by routine experiments, including but not limited to dose escalation clinical trials. When multiple therapeutic agents are used in combination, the “therapeutic effective dose” of each therapeutic agent may refer to the amount of the agent that is therapeutically effective when used alone, or to the reduced amount that is therapeutically effective when used in combination with one or more additional therapeutic agents.

[0080] Combination therapy Pharmaceutical compositions comprising either ASO or AR expression vectors disclosed herein may also be used in combination with other agents of therapeutic value in the treatment of conditions associated with pathological inflammation. In general, other agents do not necessarily need to be administered in the same pharmaceutical composition and may be administered via different routes, preferably due to their different physical and chemical properties. The determination of the mode of administration, and the appropriateness of administration in the same pharmaceutical composition, if possible, is within the scope of the knowledge of a skilled clinician. The initial administration may be carried out according to established protocols known in the art, and thereafter, based on the observed effects, the dosage, mode of administration, and timing of administration may be modified by a skilled clinician.

[0081] Compositions and pharmaceutical compositions containing ASOs and / or expression vectors, as well as additional therapeutic agents, may be administered simultaneously (e.g., simultaneously, substantially simultaneously, or sequentially within the same treatment protocol) or sequentially, depending on the stage and progression of the inflammatory disease being treated, the patient's condition, and the selection of specific therapeutic agents used. The determination of the order of administration within the treatment protocol and the number of doses of each therapeutic agent is within the scope of the knowledge of a skilled physician after evaluating the disease being treated and the patient's condition.

[0082] It is known to those skilled in the art that when used in combination therapy regimens, the effective therapeutic dose may vary. Methods for experimentally determining the effective therapeutic dose of a drug and other drugs for use in combination therapy regimens are described in the literature. For example, the use of metronomic dosing, which provides lower doses more frequently to minimize toxic side effects, is widely described in the literature. Combination therapy also includes periodic treatments that are initiated and discontinued at various times to support the clinical management of the patient.

[0083] In combination therapy, the dosage of concurrently administered therapeutic agents will, of course, vary depending on the type of concomitant drug used, the ASO or expression vector, and the disease stage of the patient being treated. The pharmaceutical compositions comprising the ASO, AR or expression vector, and additional therapeutic agents constituting the combination therapies disclosed herein may be in combined dosage forms or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical compositions constituting the combination therapy may be administered sequentially by administering one of the therapeutic agents in a regimen requiring two-step administration. A two-step administration regimen may require sequential administration of active agents or intervald administration of separate active agents. The intervals between multiple administration steps may range from several minutes to several hours, depending on the characteristics of each drug, such as potency, solubility, bioavailability, plasma half-life, and pharmacokinetic profile. Diurnal variations in various physiological parameters may also be evaluated to determine the optimal dose interval.

[0084] Examples of therapeutic agents suitable for co-administration with the compositions or pharmaceutical compositions disclosed herein include, but are not limited to, vasopressin V2 receptor antagonists (e.g., tolvaptan), angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, paracetamol, opioids, and antibiotics. [Examples]

[0085] Example 1: Identification and sequence selection of PKD1 3' UTR target sequences. Mutations in PKD1 lead to dysregulation and / or insufficient levels of functional polycystin 1 associated with the onset and severity of ADPKD. miRNAs of the miR-17 family (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) and the miR-200 family (e.g., miR-200b, miR200c, or miR-429) are known to bind to the 3' UTR of PKD1 mRNA, resulting in an increase in polycystin 1 protein.

[0086] For the 3' UTR upregulation strategy, PMOs (SEQ ID NOs) 2-351 or 353-362 are designed as part of a microwalk strategy spanning the entire PKD1 3' UTR (SEQ ID NO: 1), including binding sites for miR-17 family (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) and miR-200 family (e.g., miR-200b, miR200c, or miR-429) miRNAs. PMOs with sequences corresponding to SEQ ID NOs 47, 334, and 335 are complementary to the binding sites for miR-17 family miRNAs (Figure 1B), while PMOs with sequences corresponding to SEQ ID NOs 14, 73, 75, 336, and 337 are complementary to the binding sites for miR-200 family miRNAs (Figure 1C). miRNA binding sites are highlighted in bold (Figures 1B and 1C).

[0087] Example 2: Screening of PPMOs targeting the miR-17 binding site of PKD1 in HEK293 cells for their ability to increase PKD1 transcript expression. Peptide-PMOs (PPMOs) were generated by conjugating PMOs with sequences corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO: 47), PKD1 H46A(+611+635) (SEQ ID NO: 334), and PKD1 H46A(+615+639) (SEQ ID NO: 335) to a cell-permeable peptide (SEQ ID NO: 352). HEK293 cells were treated with PPMO or control for 24 hours, and total RNA was extracted using the MagMAX total RNA 96 extraction kit method. PKD1 gene expression was evaluated by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 transcript expression was normalized to the housekeeper TATA-binding protein (TBP, TaqMan, probe catalog number Hs00427620_m1), and the change was calculated as a fold change compared to untreated cells.

[0088] As shown in Figure 2, PPMOs corresponding to sequence numbers 47 (+604+628), 334 (+611+635), and 335 (+615+639), respectively, induced a dose-dependent increase of more than 1.1 times PKD1 mRNA compared to untreated cells. A non-targeted control (GTC CTR) predicted not to hybridize to human transcripts was included as a sham treatment. Example 3: Effect of PPMO masking the sequence in the 3' UTR of PKD1 targeted by the miR-17 miRNA seed sequence on PC1 protein levels in ADPKD patient cell lines.

[0089] For PPMO that showed significant PKD1 mRNA upregulation according to Example 2, we selected its ability to upregulate polycystin 1 (PC1) protein in ADPKD patient cell lines. ADPKD patient cell lines were established by immortalizing cells from a single proximal cortical tubular cyst isolated from an ADPKD patient with a PKD1 heterozygous mutation (p.Q2556*). PPMO was dissolved in molecular-grade H2O to prepare a 1 mM stock solution. The stock solution was diluted in culture medium and administered at 10 μM (technical replicates of n=3 per treatment condition). Untreated cells were treated with the same volume of culture medium alone. After incubating cells for 5 days, the presence of polycystin 1 (PC1) protein on the cell surface was measured by flow cytometry. Cells were incubated with anti-rabbit polycystin 1 antibody (ab74115, Abcam) and subsequently incubated with goat anti-mouse antibody conjugated to Alexa Fluor 488 (A-11001, ThermoFisher). By comparing the median fluorescence intensity (MFI) of each sample with that of untreated cells (UT), we identified PMOs that increased polycystin 1 expression, as shown in Figure 3. The PPMOs corresponding to SEQ ID NOs. 47 (+604+628), 334 (+611+635), and 335 (+615+639), respectively, induced a more than 1.1-fold increase in polycystin 1 protein compared to untreated cells.

[0090] Example 4: Functional validation of PPMO in a patient-derived primary cell 3D cyst model. PPMO, which showed significant polycystin 1 protein upregulation according to Example 3, was selected for functional validation in a patient-derived 3D cyst model. This model utilizes cystic cells extracted from kidneys provided by ADPKD patients. When grown ex vivo, these cells spontaneously form cysts in a 3D matrix and can be used to evaluate the functional effect of drug therapy on cyst growth. Immediately after seeding, patient cells were co-exposed to treatment. PPMO was dissolved in molecular-grade H2O and administered at 1 μM, 3 μM, 10 μM, and 20 μM (technical replicates of n=4 per treatment condition). Control cells were treated with the same amount of H2O alone. After 7 days of exposure, the cultures were fixed and stained for actin cytoskeleton and nucleus. Cyst growth and swelling were visualized by high-content microscopy imaging, and images were analyzed using Ominer® image analysis software. Figure 4 shows representative images of 3D patient cyst sizes after treatment with 20 μM PPMO containing sequences corresponding to sequence numbers 47(+604+628), 334(+611+635), and 335(+615+639).

[0091] Example 5: Quantification of patient-derived 3D cyst area and cell death to determine the therapeutic index. All images from the assay described in Example 4 were further analyzed, and for PPMOs having sequences corresponding to SEQ ID NOs 47(+604+628), 334(+611+635), and 335(+615+639), the cyst area (μm²) was determined. 2Dose-dependent changes in cyst growth and cell death (%) were determined. See Figure 5. PPMO corresponding to SEQ ID NO: 47 (+604+628) showed dose-dependent efficacy in inhibiting cyst growth up to 20 μM without evidence of cytotoxicity (Figures 5A and 5D). PPMO corresponding to SEQ ID NO: 334 (+611+635) effectively inhibited cyst growth, but slight cytotoxicity was observed at 20 μM (Figures 5B and 5E). PPMO corresponding to SEQ ID NO: 335 (+615+639) was found to be cytotoxic at 20 μM. However, PPMO showed moderate inhibition of cyst growth at 10 μM in a cyst model derived from ADPK patients (Figures 5C and 5F).

[0092] Example 6: Optimization of PMO sequences to enhance PKD1 upregulation by inhibiting miR-17 binding to the 3'UTR of PKD1 transcripts. PMOs (SEQ ID NOs. 353-361) were designed to microwalk SEQ ID NO. 47 by making several modifications, such as shortening the length and manipulating base mismatches. The microwalked PMOs were conjugated with a cell-permeable peptide (SEQ ID NO. 352) to produce PPMOs, and the effectiveness of PKD1 upregulation in HEK293 cells was tested. HEK293 cells were treated with PPMO or control for 24 hours. Total RNA was then extracted using the MagMAX Total RNA 96 extraction kit method. PKD1 transcript expression was evaluated by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 transcript expression was normalized against the housekeepers TATA-binding protein (TBP, TaqMan; probe catalog number Hs00427620_m1) and DexH-Box helicase 57 (DHX57, ThermoFisher; assay ID Hs00376574_m1), and the change was calculated as a fold change compared to untreated cells. The results in Figure 6 showed slight PKD1 upregulation (1.1x) when treated with PPMO(+604+626)MM16, (+604+628)MM16, (+604+626), (+604+623), (+604+628), and (+604+627).

[0093] Example 7: Screening of PPMOs to enhance PKD1 upregulation by inhibiting miR-200 binding to the 3' UTR of PKD1 transcripts. PMOs with sequences corresponding to SEQ ID NOs 14, 73, 75, and 336-337 were designed to target the binding of PKD1 mRNA to the 3' UTR and prevent miR200 binding. The designed PMOs were conjugated to a cell-permeable peptide (SEQ ID NO 352) to produce peptide-PMOs (PPMOs), and their efficacy in HEK293 cells was tested. Following 24 hours of PPMO treatment, total RNA was extracted using the MagMAX total RNA 96 extraction kit method. PKD1 gene expression was evaluated by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 transcript expression was normalized against the housekeeper TATA-binding protein (TBP, TaqMan, probe catalog number Hs00427620_m1), and the change was calculated as a fold change compared to untreated cells. As shown in Figure 7, PPMOs corresponding to SEQ ID NOs 17 (+505+529), 73 (+515+539), 336 (+677+701), and 337 (+688+712) slightly increased PKD1 mRNA expression (1.1x) compared to untreated cells.

[0094] Example 8: Evaluation of PC1 upregulation for PPMO-mediated inhibition of miR17 binding in HEK293 cells. PPMOs containing sequences corresponding to SEQ ID NOs. 47 (+604+628) and 334 (+611+635) were incubated in HEK293 cells for 5 days. The level of PC1 upregulation was evaluated using a Western blot assay. On day 5 post-treatment, proteins were extracted using RIPA buffer supplemented with a 2% protease inhibitor cocktail and 2x PhosSTOP. The protein lysates were clarified and total protein was quantified using a BCA protein kit. Samples were electrophoresed on a NuPAGE 3-8% Tris Acetate protein gel. Proteins were transferred to a nitrocellulose membrane by wet transfer. The membrane was stained for total protein and then stained with mouse anti-PC1 (Santa-Cruz, catalog no. sc130554) primary antibody, followed by anti-mouse (IRDye® 800CW preabsorbed) secondary antibody. Blots were imaged using an Odyssey Imager and quantitatively analyzed using Image Studio Ver 5.5 software (shown in Figure 8). The raw fluorescence signal of PC1 was first normalized to the raw fluorescence signal of the loading control (total protein) and then expressed as a fold change relative to UT. As shown in Figure 9, both PPMO (+604+628) and (+611+635) induced a dose-dependent increase of PC1 protein of up to 1.64 times compared to UT by day 5. Non-target controls, which were not expected to hybridize to human transcripts, were included as negative controls.

[0095] Example 9: Time-course and dose-range analysis of PPMO in PC1 protein induction in ADPKD patient-derived cell lines. WT9-7 cell lines carrying the PKD1 nonsense mutation (p.Q2556*) were incubated with PPMO (+604+628) corresponding to SEQ ID NO: 47 at concentrations of 30 μM, 60 μM, 90 μM, and 120 μM for 2, 3, and 5 days. PC1 protein levels were assessed to evaluate the efficacy of PPMO. Protein extraction was performed using RIPA buffer supplemented with a 2% protease inhibitor cocktail and 2x PhosSTOP. Protein lysates were clarified, and total protein was quantified using a BCA protein kit. Samples were electrophoresed on NuPAGE 3-8% Tris Acetate protein gel. Proteins were transferred to nitrocellulose membranes by wet transfer. Membranes were stained for total protein and then stained with mouse anti-PC1 (Santa-Cruz, catalog number sc130554) primary antibody, followed by anti-mouse (IRDye® 800CW preabsorbed) secondary antibody. Blots were imaged with an Odyssey Imager and quantitative analysis was performed using Image Studio Ver 5.5 software. The raw fluorescence signal of PC1 was first normalized to the raw fluorescence signal of the loading control (total protein) and then expressed as a magnification change relative to UT. Western blot images show PC1 expression in Figure 10. PPMO(+604+628)-SEQ ID NO: 47 induced a dose-dependent increase in PC1 protein levels up to 1.36 times compared to UT. The maximum PC1 upregulation was observed on day 2 and appeared to plateau at 90 μM.

[0096] Those skilled in the art will understand that numerous variations and / or modifications can be made to the inventions shown in specific examples without departing from the spirit or scope of the invention as broadly described. Therefore, these embodiments should be considered illustrative and non-limiting in all respects. All publications cited herein are incorporated herein in their entirety by reference. Where references are made to URLs or other such identifiers or addresses, it is understood that such identifiers may change, and certain information on the Internet may appear or disappear, but equivalent information can be found by searching the Internet. References thereto demonstrate the availability and public dissemination of such information.

[0097] Any discussion of documents, actions, materials, apparatus, articles, etc., contained herein is solely for the purpose of providing context for the present invention. It should not be construed as acknowledging that any or all of these matters formed part of the foundation of the prior art or were general knowledge in the art related to the present invention that existed prior to the priority date of each claim of this application.

[0098] References de Carvalho Ribeiro et al., 2020, Stem Cells International, doi.org / 10.1155 / 2020 / 8894590. Dugal-Tessier et al., (2021), J Clin Med., 10(4):838. Osafune et al., 2021, Clinical and Experimental Nephrology, 25(6) :574-584. Prakash et al., 2019, Nucleic Acids Research, 47(12) :6029-6044. Sharma et al., 2019, Methods Cell Biol., doi.org / 10.1016 / bs.mcb.2019.05.008

[0099] Appendix 1: Sequences and SEQ ID Nos Sequence ID 1 Human PKD1 Transcript 3' UTR (PKD-201 Standard Transcript ENST00000262304.9)

[0100] [Table 1] TIFF2026523056000002.tif254170TIFF2026523056000003.tif254170TIFF20265230560 00004.tif254170TIFF2026523056000005.tif254170TIFF2026523056000006.tif254170T IFF2026523056000007.tif254170TIFF2026523056000008.tif254170TIFF2026523056000009.tif254170TIFF2026523056000010.tif254170TIFF2026523056000011.tif23170 Sequence ID 352. Amino acid sequence of CPP RRSRTARAGRPGRNSSRPSAPR

Claims

1. An antisense oligonucleotide that binds to a target region of the 3' untranslated region (UTR) of polycystic kidney disease 1 (PKD1) mRNA, wherein the binding of the antisense oligonucleotide to the target region increases the level of polycystin 1 protein.

2. An antisense oligonucleotide that binds to a target region of the 3' untranslated region (UTR) of polycystic kidney disease 1 (PKD1) mRNA, wherein, when the binding of the antisense oligonucleotide to the target region is introduced into a 3D renal cyst culture, it reduces the growth or size of the renal cyst.

3. The antisense oligonucleotide according to claim 1 or 2, wherein the binding of the antisense oligonucleotide to the target moiety reduces the specific binding of a miR-17 family member or a miR-200 family member to the 3' UTR.

4. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the antisense oligonucleotide comprises one sequence of sequence numbers 2-351 or 353-362.

5. A vector for expressing antisense RNA (AR) in mammalian cells that binds to the target region of the 3' UTR of polycystic kidney disease 1 (PKD1) mRNA, wherein the binding of the AR to the target region reduces cyst growth when introduced into 3D renal cyst culture.

6. A vector for expressing antisense RNA (AR) in mammalian cells that binds to a target region of the 3' UTR of polycystic kidney disease 1 (PKD1) mRNA, wherein the binding of the AR to the target region increases the level of polycystin 1 protein.

7. The vector according to claim 5 or 6, wherein the binding of the AR to the target region reduces the specific binding of a miR-17 family member (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family member (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR.

8. The vector according to any one of claims 5 to 7, wherein the AR comprises one sequence of sequence numbers 2-351 or 353-362.

9. The vector according to any one of claims 6 to 8, wherein the expression vector comprises a renal cell type selective promoter for driving the expression of antisense RNA in mammalian cells.

10. The vector according to claim 9, A vector wherein the aforementioned renal cell type-selective promoter is selectively expressed in renal cells selected from the group consisting of pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending limb cells of the loop of Henle, descending limb cells of the loop of Henle, distal tubular cells, connecting tubular cells, interstitial cells, chief cells, peritubular capillary endothelial cells, and glomerular endothelial cells.

11. The vector according to any one of claims 6 to 10, wherein the vector comprises an inducible promoter.

12. The vector according to any one of claims 6 to 11, wherein the vector is a nonviral vector.

13. The vector according to any one of claims 6 to 12, wherein the vector is a viral vector.

14. The vector according to claim 13, wherein the viral vector is provided in a recombinant virus selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and anerovirus.

15. The antisense oligonucleotide or antisense RNA according to any one of claims 1 to 3, or the vector according to any one of claims 6, 7, or 9 to 14, wherein the antisense oligonucleotide or antisense RNA is bound to the target portion of the 3' UTR corresponding to SEQ ID NO:

1.

16. The antisense oligonucleotide or vector according to any one of claims 1 to 15, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the target portion over the length of the antisense oligonucleotide or antisense RNA.

17. The antisense oligonucleotide or vector according to claim 16, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA comprises one of SEQ ID NOs: 2-351 or 353-362.

18. The antisense oligonucleotide or vector according to claim 17, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA consists of one of SEQ ID NOs: 2-351 or 353-362.

19. The antisense oligonucleotide or vector according to claim 17 or claim 18, wherein the nucleotide sequence of the antisense oligonucleotide includes or consists of one of SEQ ID NOs: 17, 47, 73, 334-337, or 355-360.

20. The antisense oligonucleotide or vector according to claim 19, wherein the nucleotide sequence comprises or consists of either SEQ ID NO: 334 or 335.

21. The antisense oligonucleotide or vector according to claim 19, wherein the nucleotide sequence comprises or consists of any one of sequence numbers 355-360.

22. The antisense oligonucleotide or vector according to claim 19, wherein the nucleotide sequence comprises or consists of any one of SEQ ID NOs: 17, 73, 336, and 337.

23. The antisense oligonucleotide or vector according to claim 19, wherein the nucleotide sequence includes or consists of SEQ ID NO:

47.

24. The antisense oligonucleotide according to any one of claims 1 to 3 or 15 to 23, wherein the antisense oligonucleotide includes a skeletal modification.

25. The antisense oligonucleotide according to claim 24, wherein the skeletal modification includes a phosphorothioate bond or a phosphorodiamidate bond.

26. The antisense oligonucleotide according to claim 24 or 25, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, or a 2'-O-modification such as a 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

27. The antisense oligonucleotide according to any one of claims 24 to 26, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

28. The antisense oligonucleotide according to claim 27, wherein each sugar portion in the antisense oligonucleotide is a modified sugar portion.

29. The antisense oligonucleotide according to any one of claims 24 to 26, wherein the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

30. The antisense oligonucleotide according to claim 29, wherein each nucleotide of the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

31. The antisense oligonucleotide or vector according to any one of claims 1 to 30, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA consists of 20 to 30 nucleotides, 22 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, or 26 to 30 nucleotides.

32. The antisense oligonucleotide or vector according to claim 31, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA consists of 25 to 30 nucleotides.

33. The antisense oligonucleotide according to claim 32, wherein the antisense oligonucleotide comprises one or more phosphorodiamidate morpholino moieties.

34. The antisense oligonucleotide according to any one of claims 1 to 4 or 15 to 33, further comprising a functional portion to which the antisense oligonucleotide is linked.

35. The antisense oligonucleotide according to claim 34, wherein the functional portion includes a delivery portion.

36. The antisense oligonucleotide according to claim 35, wherein the delivery portion is selected from the group consisting of lipids, peptides, polyethers, carbohydrates, and antibodies.

37. The antisense oligonucleotide according to claim 35 or claim 36, wherein the delivery portion comprises a cell-permeable peptide (CPP).

38. The antisense oligonucleotide according to claim 37, wherein the amino acid sequence of the CPP includes or consists of SEQ ID NO:

352.

39. The antisense oligonucleotide according to claim 35 or claim 36, wherein the delivery portion comprises a receptor-binding domain (RBD).

40. The antisense oligonucleotide according to any one of claims 35 to 39, wherein the delivery portion comprises an N-acetylgalactosamine (GalNAc) portion, a poly(ethylene glycol) (PEG) portion, a fatty acid portion, or a lipid portion.

41. The antisense oligonucleotide according to claim 34, wherein the functional portion includes a stabilizing portion.

42. The antisense oligonucleotide according to any one of claims 34 to 41, wherein the functional portion is covalently bonded to the antisense oligonucleotide.

43. The antisense oligonucleotide according to any one of claims 34 to 41, wherein the functional portion is non-covalently bonded to the antisense oligonucleotide.

44. The antisense oligonucleotide according to any one of claims 34 to 41, wherein the functional portion is linked to the 5' end of the antisense oligonucleotide.

45. The antisense oligonucleotide according to any one of claims 34 to 41, wherein the functional portion is linked to the 3' end of the antisense oligonucleotide.

46. A pharmaceutical composition comprising an antisense oligonucleotide or vector according to any one of claims 1 to 45 and a pharmaceutically acceptable excipient.

47. A method for treating autosomal dominant polycystic kidney disease (ADPKD), comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 46 to a subject in need thereof.

48. The method according to claim 47, wherein the subject is a human subject.

49. The method according to claim 47 or claim 48, wherein the growth or size of a renal cyst is reduced.

50. Use of an antisense oligonucleotide or vector according to any one of claims 1 to 45 in the manufacture of a pharmaceutical product for the treatment of ADPKD.

51. A method for increasing intracellular polycystin 1 protein ex vivo or in vivo, comprising contacting a cell or tissue with an antisense oligonucleotide or vector according to any one of claims 1 to 45, or a pharmaceutical composition according to claim 42.

52. The method according to claim 51, wherein the cells are located within a 3D human renal cyst culture.

53. Genetically modified cells comprising an antisense oligonucleotide or vector according to any one of claims 1 to 41.

54. The gene-modified cell according to claim 53, wherein the gene-modified cell is a mammalian cell.

55. The genetically modified mammalian cell according to claim 54, wherein the genetically modified mammalian cell is a human cell.

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