Compositions and methods for treating kidney disease

Abstract

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

Description

[0001] This application claims priority to AU 2023901907 filed 16 June 2023 and AU 2023903042 filed 21 September 2023, the entire contents of each of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates generally to oligonucleotides for treating a condition associated with a mutation in the polycystic kidney disease 1 (PKD1) gene, and related compositions and methods. PKD1 BACKGROUND BACKGROUND

[0003] Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease leading to end-stage renal disease (ESRD). Approximately 1 in 500-2,500 individuals carry a mutation for this condition. ADPKD is a progressive disorder characterized by abnormal dilation of tubular cells, leading to multiple cysts in the kidney.

[0004] Cyst development begins early in life, and in more severe cases, in utero, and is often followed by a long period of asymptomatic progression. 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 abnormally rapid progressive disease. In line with declining kidney function, patients develop various urological complications, such as cysts and urinary tract infections, decreased glomerular filtration rate, chronic lower back pain, and hypertension. ADPKD patients often develop additional extra-renal conditions, including liver cysts (>90% of patients >35 years of age), pancreatic cysts, intracranial aneurysms, colonic diverticular disease, and heart valve defects. Overall, ADPKD is associated with significant morbidity, shortened life expectancy.

[0005] ADPKD is primarily caused by mutations in one of the following polycystin genes: PKD1 polycystin 1, transient receptor potential channel-interacting (74-85% of patients) or PKD2 (15-26%). Patients with mutations in PKD2 generally have a milder phenotype than patients with mutations in PKD1, which is reflected in a difference of approximately 20 years in the average age of ESRD (PKD1 disease patients at 54.3 years, versus PKD2 disease patients at 74.0 years). PKD2 PKD1 There is an ongoing need for new treatments or preventative measures for ADPKD. SUMMARY

[0006]

[0007] PKD1 ​​It consists of 46 exons of approximately 50 kb of genomic DNA spanning the short arm of chromosome 16 (16p13.3). (Canonical) PKD1 The encoded polycystin 1 protein is a 4,303-amino acid glycosylated membrane-integrated protein localized to primary cilia, endoplasmic reticulum, adhesion and desmosome junctions, apical membrane, plasma membrane, and junctional complexes. Polycystin 1 contains a large N-terminal extracellular domain, multiple transmembrane domains, and a cytoplasmic C-tail, and acts as a regulator of calcium-permeable cation channels and 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 isotypes of this gene have been noted.

[0008] While not wishing to be bound by theory, it is believed that kidney cysts develop in ADPKD once polycystin 1 function falls below a certain level. Indeed, this is consistent with the fact that the level of residual polycystin 1 function from mutated genes is directly correlated with disease severity. The median age of onset for ESRD was 55 years in carriers of truncated mutations (complete loss of function) and 67 years in carriers of non-truncated mutations (partial loss of function).

[0009] This disclosure provides antisense oligonucleotides (ASO), antisense RNA (AR) expression vectors, and related compositions and methods for regulating... PKD1 Post-transcriptional or post-translational regulation of the 3' untranslated region (UTR) of 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 homologous binding sites to enhance [the regulation of the mRNA]. PKD1 mRNA and / or polycystin 1 protein levels.

[0010] Therefore, on the one hand, this paper provides an ASO that is compatible with... PKD1 The target portion of the 3' UTR of mRNA binds to the antisense oligonucleotide, wherein the binding of the antisense oligonucleotide to the target portion increases the level of polycystic kidney disease 1 (PCD1). On the other hand, this paper provides an ASO that is associated with PCD1 (PCD1). PKD1) a targeted portion of a 3' untranslated region (UTR) of mRNA, wherein binding of the antisense oligonucleotide to the targeted portion reduces kidney cyst growth or reduces kidney cyst size when introduced into a 3D kidney cyst culture. In some examples, binding of the antisense oligonucleotide to the targeted portion reduces specific binding of a miR-17 family miRNA (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family miRNA (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR. In some examples, wherein binding of the antisense oligonucleotide to the targeted portion reduces specific binding of a mirR-17 family miRNA (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family miRNA (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR, the antisense oligonucleotide comprises the sequence of any one of SEQ ID NOs: 2-351 or 353-362.

[0011] In another aspect, provided herein is a vector for expressing an AR that binds to a targeted portion of a 3' UTR of mRNA in a mammalian cell, wherein the AR binds to the targeted portion of the 3' UTR of mRNA. PKD1 In another aspect, provided herein is a vector for expressing an AR that binds to a targeted portion of a 3' UTR of mRNA in a mammalian cell, wherein the AR binds to the targeted portion of the 3' UTR of mRNA. PKD1a targeted portion of a 3' UTR of an mRNA, wherein binding of the AR to the targeted portion reduces cyst growth when introduced into a 3D kidney cyst culture. In some examples, the binding of the AR to the targeted portion reduces specific binding of a mirR-17 family miRNA (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family miRNA (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR. In some examples, wherein the binding of the AR to the targeted portion reduces specific binding of a mirR-17 family miRNA (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family miRNA (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR, the AR comprises or consists of the sequence of any of any 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, wherein the vector is a viral vector, the viral vector is provided in the form of a recombinant virus selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, and an anellovirus.

[0012] In some examples, the AR can be delivered by a vector (e.g., a plasmid or a recombinant virus) comprising a kidney cell type-selective or tissue-selective promoter for driving expression of the AR in the mammalian cell. In some examples, the promoter is selective for expression in a kidney cell selected from the group consisting of a pericyte, a podocyte, a parietal epithelial cell, a proximal tubule cell, an ascending loop of Henle cell, a descending loop of Henle cell, a distal tubule cell, a juxtaglomerular cell, an intercalated cell, a principal cell, a peritubular capillary endothelial cell, and a glomerular endothelial cell. In some examples, the vector comprises an inducible promoter.

[0013] In some examples, in any of the foregoing ASOs or vectors, the ASO or the AR binds within a targeted 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 targeted portion over the length of the ASO or the AR. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of any of SEQ ID NOS: 2-351 or 353-362. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of any of SEQ ID NOS: 17, 47, 73, 334-337, or 355-360. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of any of SEQ ID NOS: 334 or 335. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of any of SEQ ID NOS: 355-360. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of any of SEQ ID NOS: 17, 73, 336, and 337. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of SEQ ID NO: 47.

[0014] In some examples, any of the foregoing ASOs include a backbone modification. In some examples, the backbone modification includes a phosphorothioate or phosphorodiamidate linkage. In other examples, the ASO includes a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, or 2'-0- O - modified sugar moiety, such as a 2'-0- O - methyl, 2'-fluoro, or 2'-0- O - methoxyethyl moiety. In some examples, the ASO includes at least one modified sugar moiety. In other examples, each sugar moiety in the ASO is a modified sugar moiety. In some examples, the ASO includes a 2'-0- O - methoxyethyl moiety. In other examples, each nucleotide of the ASO includes a 2'-0- O - methoxyethyl moiety.

[0015] In some examples of any of the foregoing ASOs or vectors, the nucleotide sequence of the ASO or AR consists of 20 to 30 nucleotides, 22 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, or 26 to 30 nucleotides. In some examples, the nucleotide sequence of the ASO or the AR consists of 25 to 30 nucleotides. In some examples, wherein the sequence of the ASO consists of 20 to 30 nucleotides, the ASO comprises one or more phosphorodiamidate morpholino moieties.

[0016] In some examples, any of the foregoing ASOs further comprises a linked functional moiety. In some examples, the functional moiety comprises a delivery moiety. In some examples, the delivery moiety is selected from the group consisting of a lipid, a peptide, a carbohydrate, a polyether (e.g., polyethylene glycol), and an antibody. In some examples, wherein the ASO comprises a delivery moiety, the delivery moiety comprises a cell penetrating peptide (CPP). In some examples, the delivery moiety comprises a receptor binding domain (RBD). In some examples, the delivery moiety comprises a poly(ethylene glycol) (PEG) moiety. In some examples, the delivery moiety comprises N an acetylgalactosamine (GalNAc) moiety. In some examples, the delivery moiety comprises a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is about C8 to C20. In other examples, the functional moiety comprises a stabilization moiety. In some examples, the functional moiety is covalently linked to the ASO. In other examples, the functional moiety is non-covalently linked 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.

[0017] In a related aspect, provided herein is a pharmaceutical composition comprising any of the foregoing ASOs or vectors, and a pharmaceutically acceptable excipient.

[0018] In a further related aspect, provided herein is a method for treating autosomal ADPKD, wherein a therapeutically effective amount of the foregoing pharmaceutical composition is administered to a subject in need thereof. In some examples, the subject to be treated is a human subject. In another related aspect, provided herein is use of any of the foregoing ASOs, vectors for the preparation of a medicament for the treatment of ADPKD.

[0019] In another aspect, provided herein is a method of increasing PKD1 mRNA and / or polycystin-1 protein in an ex vivo cell or in vivo tissue, wherein the method comprises the step of contacting the cell or tissue with any of the foregoing ASOs, vectors, or pharmaceutical compositions.

[0020] In yet another aspect, provided herein is a genetically modified cell comprising any of the foregoing ASOs or vectors. In some examples, the genetically modified cell is a mammalian cell. In some examples, the genetically modified mammalian cell is a genetically modified human cell. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 - PKD1 Exon-intron structure, PKD schematic representation of miRNA binding sites in the 3' UTR and relative binding position of exemplary PMOs. (A) Exon-intron map of the human PKD1 pre-mRNA; (B) PKD1 sequence of the mRNA 3' UTR, including binding sites for 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), respectively, are indicated; (C) PKD1 sequence of the mRNA 3' UTR, including binding sites for miR-200 family miRNAs (bold). Relative positions of complementary PMOs with sequences corresponding to SEQ ID NOs: 14 (+505+529), 337 (+515+539), 336 (+677+701), 73 (+682+706), and 75 (+688+712), respectively, are indicated.

[0022] Figure 2 - Screening of PPMOs targeting miR-17 binding sites in HEK293 cells. PKD1 Figure 2 - Screening of PPMOs targeting miR-17 binding sites in HEK293 cells.

[0023] PPMOs with sequences corresponding to SEQ ID NOs: 47 (+604+628), 334 (+611+635), and 335 (+615+639) were screened in HEK293 cells. Graphs show PKD1 mRNA expression at 24 hours after treatment with PPMOs targeting the miR-17 site or a non-targeting control (GTCCTR). mRNA expression was normalized to PKD1 mRNA expression and expression in untreated cells was set to 1. TBP

[0024] Figure 3 - Effect of PPMOs targeting sequences in the 3' UTR of PKD1 that bind to the miR-17 miRNA seed sequence in ADPKD patient cell lines. ​

[0025] According to Example 2, PPMOs showing significant PKD1 PPMOs capable of upregulating polycystin 1 (PC1) protein in a cell line from an ADPKD patient carrying a PKD1 heterozygous mutation (p.Q2556*) mRNA upregulation. Graph showing the median fluorescence intensity (MFI) of PC1 protein staining on the cell surface measured by flow cytometry after 5 days of treatment with 10 µM miR-17 site-targeting PPMOs. Polycystin staining MFI was normalized to the antibody control of isotype staining, and the MFI in untreated cells was set to 1.

[0026] Figure 4 - Functional validation of PPMOs in patient-derived 3D cyst models.

[0027] Patient-derived cyst cells were functionally validated with PPMOs (PPMOs) having sequences corresponding to SEQ ID NOs: 47 (+604+628), 334 (+611+635), and 335 (+615+639). PPMOs were dosed at 1 µM, 3 µM, 10 µM, and 20 µM, and after 7 days of exposure, cultures were fixed and stained for actin cytoskeleton and nuclei. Growth and swelling of cysts were visualized by high-content microscopy imaging. Representative images show cyst size in wells treated with 20 µM PPMO 7 days after treatment. Assay was performed under unstimulated conditions (spontaneous cyst formation).

[0028] Figure 5 - Quantification of patient-derived 3D cyst area and cell death.

[0029] Images from all assay conditions described in Figure 4 were further analyzed to determine dose-dependent changes in cyst area (µm 2 ) and cell death (%), to distinguish efficacy from cytotoxicity. A cutoff value for cell death was set at 15%.

[0030] (A-C) Swelling inhibition was determined by measuring cyst area. This was calculated as the average area of each cyst in each plane of the z-stack. (D-F) Cytotoxicity was calculated as the proportion of dead cells. Cells were scored as ‘dead’ if their nuclei were unassociated with colocalized actin cytoskeleton (rhodamine phalloidin labeled). This value was normalized to the solvent control (0%) and expressed as mean + / - standard deviation of replicates.

[0031] Figure 6: PPMO micro-walk screening for inhibition of miR-17 binding PPMOs corresponding to the oligonucleotide sequences in SEQ ID NOs: 353-361 were incubated with HEK293 cells at concentrations of 3 µM, 10 µM, and 30 µM for 24 hours, with n = 3 technical replicates per treatment condition. A non-targeting control PPMO designed to not hybridize to any known human transcript was used as a negative control treatment. This control PPMO was conjugated to the same cell-penetrating peptide as the test PPMOs. A positive control oligonucleotide (RGLS4326, Med Chem Express, Catalog No. HY-139290), an inhibitor of miR-17, was included as an assay control. Data were normalized to two housekeeping genes and reported as relative levels compared to untreated cells set as 1. Data represent mean ± S.D., and UT = untreated cells. n = 1 biological replicate. TBP = TATA binding protein, DHX57 = DExH-box helicase 57.

[0032] Figure 7: Screening PPMOs that inhibit miR-200 binding PPMOs corresponding to the oligonucleotide sequences in SEQ ID NOs: 14, 73, 75, 336-337 were incubated with HEK293 cells at concentrations of 3 µM, 10 µM, and 30 µM for 24 hours, with n = 3 technical replicates per treatment condition. A non-targeting control PPMO designed to not hybridize to any known human transcript was used as a negative control treatment. This control PPMO was conjugated to the same cell-penetrating peptide as the test PPMOs. A positive control oligonucleotide (RGLS4326, Med Chem Express, Catalog No. HY-139290), an inhibitor of miR-17, was also included. Data were normalized to two housekeeping genes and reported as relative levels compared to untreated cells set as 1. Data represent mean ± S.D., and UT = untreated cells. n = 2 biological replicates. TBP = TATA binding protein, DHX57 = DExH-box helicase 57.

[0033] Figure 8: Western blot images of PPMO-treated HEK293 cells HEK293 cells were treated with PPMO (+604+628) and (+611+635) having sequences corresponding to SEQ ID NO: 47 and 334, respectively. Non-targeting control (NTC) was used as a negative control, while an inhibitor of miR-17 (RGLS4326, Med Chem Express, Cat# HY-139290) was included as a positive control. PPMO-treated cells were harvested at day 5 and PC1 expression was analyzed using Western blotting. Two protein bands of approximately 462 kDa and 350 kDa indicate full-length (FL) and N-terminal fragment (NTF) of PC1, respectively. The intensity of total protein staining was analyzed as a loading control. Experiments were performed in quadruplicate.

[0034] Figure 9: Quantification of PC1 protein upregulation in PPMO-treated HEK293 cells In Figure 8, bar graphs represent the mean + S.D. of PC1 protein expression using gel image analysis. Both FL and NTF bands were included in the analysis and normalized to total protein staining. Dotted line indicates untreated baseline PC1 protein level set to 1. Experiments were performed in quadruplicate. n = 2 biological replicates. UT = untreated cells. NTC = non-targeting control.

[0035] Figure 10: PC1 protein analysis in PPMO-treated WT9-7 cells WT9-7 cells were incubated with PPMO having sequence corresponding to SEQ ID NO: 47 (+604+628) and non-targeting control (NTC) in quadruplicate for 2, 3 or 5 days. Non-targeting control (NTC) was used as a negative control and an inhibitor of miR-17 (RGLS4326, Med Chem Express, Cat# HY-139290) was included as a positive control. Bar graphs represent the mean + S.D. of PC1 protein normalized to total protein staining relative to untreated cells. n = 1 biological replicate. UT = untreated cells. NTC = non-targeting control. DETAILED DESCRIPTION

[0036] Review Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly indicates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth.

[0037] Each example of the disclosure described herein is adapted to apply mutatis mutandis to each of the other examples, unless specifically stated otherwise.

[0038] Those skilled in the art will appreciate that, except for the specifically described disclosure, changes and modifications can be made to the disclosure herein. It will be understood that the disclosure includes all such changes and modifications. The disclosure also includes all steps, features, compositions and compounds individually or collectively referred to or indicated in the specification, as well as any and all combinations of any two or more of the steps or features.

[0039] The disclosure is not limited to the specific examples described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods, as described herein, are clearly within the scope of the disclosure.

[0040] The present disclosure is conducted without undue experimentation using conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis and immunology, unless otherwise specifically indicated. Such techniques are described and explained throughout the literature in 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 thereto, Coligan et al. (eds) (including all updates thereto), Maniatis et al. 1982, Gait (ed.) 1984, Hames and Higgins (eds) 1984, Freshney (ed.) 1986.

[0041] The term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both alternatives (or either alternative) conjunctive or disjunctive meanings.

[0042] The term "about" means + / - 20%, more preferably + / - 10% of the indicated value, unless stated otherwise. For the avoidance of doubt, the term "about", followed by an indicated value, is to be interpreted also to include the exact indicated value itself (e.g. "about 10" is to be interpreted as also exactly 10).

[0043] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0044] As used herein, the term "antisense oligonucleotide", "antisense oligomer" or "ASO" encompasses oligonucleotides and any other oligomer molecule comprising nucleobases capable of hybridizing to a complementary sequence on a target RNA transcript, including but not limited to those that do not comprise sugar moieties, as in the case of peptide nucleic acids (PNAs). Preferably, the ASO is a nuclease-resistant cleavage or degradation-resistant ASO.

[0045] As used herein, the phrase "binds to" or "binds within" a targeted portion with respect to an ASO or AR refers to specific hybridization between the ASO or AR nucleotide sequence and a target nucleotide sequence that is complementary within the ranges set forth herein. In some examples, specific hybridization occurs under conditions where hybridization occurs under high stringency conditions. "High stringency conditions" means that under such in vitro conditions, the ASO or AR hybridizes to the target sequence, with a detectably greater amount of specificity than to non-specific sequences by at least about 10% as compared to a non-specific sequence. High stringency conditions are then conditions that distinguish between a polynucleotide with an exact complementary sequence or a polynucleotide containing only a few scattered mismatches from a random sequence that happens to have a few small regions (e.g., 1-5 bases) that match the probe. Such a region of complementarity is more easily melted than a full-length complement of 12-17 or more bases, and moderate stringency hybridization makes it easy to distinguish. In one example, high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or equivalent ionic strength, at temperatures of about 50-70 °C. The skilled artisan will understand that under in vivo conditions, the specificity of hybridization between an ASO or AR and its target sequence is defined by the level of complementarity between the ASO or AR and the target sequence to which it hybridizes within the cell.

[0046] The term "peptide" is intended to include compounds composed of amino acid residues joined by amide bonds. The peptide can be natural or non-natural, ribosomally encoded or synthetically derived. Typically, a peptide will consist of 2 to 200 amino acids. For example, a peptide can range in length from 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 ranges recited. A peptide can comprise or consist of fewer than about 150 amino acids, or fewer than about 125 amino acids, or fewer than about 100 amino acids, or fewer than about 90 amino acids, or fewer than about 80 amino acids, or fewer than about 70 amino acids, or fewer than about 60 amino acids, or fewer than about 50 amino acids.

[0047] Peptides as referred to herein include "inverso" peptides in which all L-amino acids are replaced by the corresponding D-amino acids; "retro-inverso" peptides in which the amino acid sequence is reversed and all L-amino acids are replaced by D-amino acids.

[0048] A peptide can comprise amino acids in both L- and / or D-forms. For example, both L- and D-forms can be used for different amino acids within the same peptide sequence. In some examples, the amino acids within the peptide sequence are in L-form, as natural amino acids. In some examples, the amino acids within the peptide sequence are a combination of L- and D-forms. Further, a peptide can comprise unusual but naturally occurring amino acids, including but not limited to hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr). A peptide can also incorporate non-natural amino acids, including but not limited to homoamino acids, N-methyl amino acids, alpha-methyl amino acids, beta (homo) amino acids, gamma amino acids, and N-substituted glycines. A peptide can be a linear peptide or a cyclic peptide.

[0049] The term "protein" shall be taken to include a single polypeptide chain, i.e. a series of contiguous amino acids joined by peptide bonds, or a series of polypeptide chains (i.e. a polypeptide complex) covalently or non-covalently linked to one another. For example, the series of polypeptide chains can be covalently linked using suitable chemical bonds or disulphide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces and hydrophobic interactions.

[0050] The percent amino acid sequence identity with respect to a given amino acid sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any 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 Institute (EMBL-EBI) as part of the European Molecular Biology Laboratory. The tool is available on the website located at www.ebi.ac.uk / Tools / emboss / align / . The tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). With default settings, which include gap opening: 10.0 and gap extension 0.5. The default matrix "Blosum62" is used for amino acid sequences and the default matrix.

[0051] The term "cell-penetrating peptide" (CPP) refers to a peptide that is capable of crossing a cell membrane. In one example, a CPP is capable of translocating across a mammalian cell membrane and into a cell. In another example, a CPP can direct a conjugate to a desired subcellular compartment. Thus, a CPP can direct or facilitate the penetration of a molecule of interest through a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A CPP can translocate across a membrane in its entirety and intact or alternatively, be partially degraded.

[0052] A CPP can direct a molecule of interest, such as an ASO as disclosed herein, from outside a cell through the plasma membrane and into the cytoplasm or a desired subcellular compartment. Alternatively or additionally, a CPP can direct a molecule of interest through an epithelial barrier, an endothelial barrier, a basement membrane barrier, a transmucosal barrier, a cardiovascular barrier, a skin barrier, a gastrointestinal barrier, and / or a lung barrier. In some embodiments, a CPP selectively targets or is taken up by the kidney.

[0053] The term "peptide ligand" or "receptor binding domain" refers to a peptide that is capable of binding to a membrane surface receptor to enable the peptide to translocate across the cell membrane. In one example, the peptide ligand can effect translocation across the cell membrane by targeting the natural endocytosis of the receptor. In another example, the peptide ligand can utilize a complementary mechanism of translocation across the cell membrane, including by utilizing a conjugated CPP. In one example, the peptide ligand is capable of translocating across a mammalian cell membrane and into a cell. In another example, the peptide ligand can direct the conjugate to a desired subcellular compartment. Thus, the peptide ligand can direct or facilitate cellular uptake of a molecule of interest through a phospholipid, mitochondria, endosome, lysosome, vesicle, or nuclear membrane. The peptide ligand can translocate across the membrane entirely and intact or alternatively be partially degraded.

[0054] The binding of the peptide ligand to the target receptor can direct the molecule of interest, such as an ASO as disclosed herein, from outside the cell through the plasma membrane and into the cytoplasm or a desired subcellular compartment. Alternatively or additionally, the binding of the peptide ligand to the target receptor can direct the molecule of interest through a relevant biological barrier, such as a kidney basement membrane barrier, a blood-brain barrier, a transmucosal barrier, a blood-retinal barrier, a cardiovascular barrier, a skin barrier, a gastrointestinal barrier, and / or a lung barrier.

[0055] Compositions for increasing PKD1 mRNA and polycystin-1 protein levels MicroRNAs (miRNAs) are a family of short (19-23 nucleotides) non-coding single-stranded labile RNAs. Although it can bind to any part of the target mRNA, its main mode of action is to bind to complementary RNA sequences in the 3' untranslated region (3' UTR) and modulate gene expression by stimulating mRNA degradation or translational repression. Both mechanisms result in decreased expression of the target gene. In the case of the polycystic kidney disease 1 (PKD1) gene, which encodes the polycystin 1 protein, miRNAs that bind to the 3' UTR and the coding 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). PKD1

[0056] While not wishing to be bound by theory, it is believed that an antisense sequence that is at least partially complementary to the microRNA binding site located within the mRNA 3' UTR will hybridize to the 3' UTR and sterically hinder ("mask") these microRNAs from accessing their binding sites in the 3' UTR. PKD1 The hybridization of the antisense sequence to the 3' UTR is believed to at least partially block the binding of the microRNAs to the 3' UTR and, in turn, to the coding mRNA transcript. PKD1 3' UTR, and in turn, to the coding mRNA transcript. PKD1 ​3' UTR, ultimately resulting in increased levels of polycystin 1 protein. Accordingly, disclosed herein is an ASO that binds to PKD1 a targeted portion of the 3' UTR of a mRNA, wherein binding of the antisense oligonucleotide to the targeted portion increases PKD1 the level of the mRNA and / or polycystin 1 protein. In some preferred examples, the ASO hybridizes to PKD1 a targeted portion of the 3' UTR of a mRNA, whereby one or more miRNAs are unable to hybridize to their specific target sequences and, as a result, the level of the mRNA and polycystin 1 protein is increased. PKD1 the mRNA and polycystin 1 protein is increased.

[0057] Also disclosed herein is a vector for expressing an AR in a mammalian cell that binds to PKD1 a targeted portion of the 3' UTR of a mRNA, ultimately resulting in increased levels of polycystin 1 protein as described above.

[0058] For reference, the canonical human PKD1 mRNA transcript ("PKD-201") sequence is publicly available through the online Ensembl database, accession ENST00000262304.9. The canonical human PKD1 mRNA 3' UTR sequence is provided herein as SEQ ID NO: 1.

[0059] Antisense oligonucleotides (ASO) and antisense RNA (AR) In some preferred examples of the compositions and methods described herein, the ASO and AR have a sequence that is fully complementary or near-complementary (e.g., sufficient complementarity to bind to the target sequence and interfere with PKD1 miRNA binding at the mRNA 3' UTR binding site) across their length to the target sequence. The ASO and AR are designed such that they bind (hybridize) to the target RNA sequence (e.g., the targeted portion of the pre-mRNA transcript) and remain hybridized under physiological conditions. Where possible, the appropriate sequence is selected for the ASO and AR to generally avoid similar nucleic acid sequences in other (i.e., off-target) locations in the genome or cellular mRNA or miRNA such that the likelihood of the ASO or AR hybridizing at these sites is limited.

[0060] In some examples, the ASO or AR "specifically hybridizes" or has "specificity" for the targeted portion of the target nucleic acid or PKD1 mRNA 3' UTR. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence is hybridized to the complementary oligonucleotide.

[0061] When hybridization occurs between two single-stranded polynucleotides of complementary sequences in an antiparallel configuration, the ASO and AR sequences are “complementary” to their target sequences. Complementarity can be quantified according to generally accepted base pairing rules based on the proportion (e.g., percentage) of bases in the opposite strands expected to form hydrogen bonds with each other. The nucleotide sequence of the ASO or AR does not need to be 100% complementary to the nucleotide sequence of the target nucleic acid it hybridizes with. In some instances, the nucleotide sequence of the ASO or AR in the compositions disclosed herein may be complementary to 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% of the nucleotide sequence of the targeted portion of the RNA transcript in length of the ASO or AR nucleotide sequence. For example, an ASO or AR with 18 out of 20 nucleotides complementary to the target region and thus specifically hybridizing would represent 90% complementarity. In such instances, the remaining non-complementary nucleotides of the ASO or AR may be clustered together or interspersed with complementary nucleotides and do not need to be continuous. The complementarity of the ASO or AR sequence to the target nucleotide sequence (expressed as a "percentage of complementarity" with its target sequence; or a "percentage of identity" with its anticomplementary sequence) can be routinely determined using algorithms known in the art, such as the BLAST procedure (Basic Local Alignment Search Tool) and the PowerBLAST procedure (Altschul et al., 1990, Journal of Molecular Biology). J. Mol. Biol .)》, 215:403-410; Zhang et al., 1997, "Genome Research ( Genome Res .)》, 7:649-656).

[0062] In some instances, ASO or AR does not hybridize with all nucleotides in the target sequence, and the positions of the hybridized nucleotides can be continuous or discontinuous. ASO or AR can hybridize on one or more segments of the 3' UTR of the mRNA, such that segments in the middle or adjacent are not involved in hybridization events (e.g., loop structures or hairpin structures can be formed).

[0063] In some instances, the nucleotide sequences of the ASO or AR described herein are similar to... PKD1 The targeted portion of the mRNA 3' UTR is complementary. In some preferred examples, ASO or AR corresponds to SEQ ID NO: 1. PKD1 The targeted portion of the mRNA 3' UTR is complementary. In some instances, the nucleotide sequence of the ASO or AR is similar in length to that of the ASO or AR. PKD1The nucleotide sequence of the targeted portion of the 3' UTR is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some examples, the nucleotide sequence of the ASO or AR has 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 across the full length of any of the sequences provided in SEQ ID NOs: 2-351 or 353-362 (as shown in Table 1).

[0064] The ASOs or ARs for use in the compositions described herein can have any length suitable for specific hybridization with a 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 can 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 consists of more than 50 nucleotides but no more than 100 nucleotides in length. In some examples, the ASO or AR nucleotide sequence is 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 35 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 9 to 50 nucleotides, 9 to 40 nucleotides, 9 to 35 nucleotides, 9 to 30 nucleotides, 9 to 25 nucleotides, 9 to 20 nucleotides, 9 to 15 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, 10 to 15 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 11 to 20 nucleotides, 11 to 15 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 35 nucleotides, 12 to 30 nucleotides, 12 to 25 nucleotides, 12 to 20 nucleotides, 12 to 15 nucleotides, 13 to 50 nucleotides, 13 to 40 nucleotides, 13 to 35 nucleotides, 13 to 30 nucleotides, 13 to 25 nucleotides, 13 to 20 nucleotides, 14 to 50 nucleotides, 14 to 40 nucleotides, 14 to 35 nucleotides, 14 to 30 nucleotides, 14 to 25 nucleotides, 14 to 20 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 15 to 20 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 20 to 25 nucleotides, 25 to 50 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 25 to 30 nucleotides in length. In some examples, the ASO or AR is 20 nucleotides in length. In some preferred examples, the nucleotide sequence of the ASO or AR nucleotide is 25 nucleotides in length.

[0065] In some examples of each occurrence of “G” in the ASO or AR sequences disclosed herein, “G” is guanosine or inosine. In some examples of each occurrence of “T” in the ASO or AR sequences disclosed herein, “T” is any one of: thymidine, inosine, uracil, or an isomer or modified form of uracil (e.g., pseudouridine or N1-methyl-pseudouridine). In some examples of 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).

[0066] In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one of SEQ ID NOs: 2-351 or 353-362. In some examples, the nucleotide sequence of the ASO or AR consists of the sequence of any one of SEQ ID NOs: 2-351 or 353-362. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of the sequence of any one of SEQ ID NOs: 17, 47, 73, 334-337, or 355-360. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of the sequence of any one of SEQ ID NOs: 334 or 335. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of the sequence of any one of SEQ ID NOs: 355-360. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of the sequence of any one of SEQ ID NOs: 17, 73, 336, and 337. In some examples, the nucleotide sequence of the ASO or AR comprises or consists of the sequence of SEQ ID NO: 47.

[0067] ASO chemistries and modifications The ASOs used in the compositions described herein can comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination thereof. The term “naturally occurring nucleotide” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotide” includes nucleotides having a modified or substituted sugar group and / or having a modified backbone. In some examples, all of the nucleotides of an ASO are modified nucleotides. Chemical modifications of ASOs or ASO components compatible with the compositions and methods described herein are known in the art, as disclosed in, e.g., U.S. Patent No. 8,258,109, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Roberts et al., 2020, Nature Reviews Drug Discovery, 19:673-694. Nature Rev. Drug Disc. )

[0068] The one or more nucleotides of an ASO can be any naturally occurring, unmodified nucleobase (such as adenine, guanine, cytosine, thymine, uracil, and inosine) or any synthetic or modified nucleobase that is classifiable as such, such that it is capable of hydrogen bonding with a nucleobase present on the target RNA transcript. Examples of suitable modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0069] An ASO includes a “backbone” structure, which refers to the linkage between the nucleotides / monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises 3’-5’ phosphodiester bonds linking the sugar moieties of adjacent nucleotides. Types of backbone linkages suitable for use in the ASOs described herein include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphorodiamidate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanilide, phosphoramidate, and the like. In some examples, the backbone modification is a phosphorothioate linkage. In other examples, the backbone modification is a phosphorodiamidate linkage. See, e.g., Roberts et al., supra; and Agrawal (2021), Biomolecules, 9:503. In some examples, the backbone structure of the ASO does not contain phosphorus-based linkages, but rather contains, for example, peptide linkages in peptide nucleic acids (PNAs) or linking groups that include carbamates, amides, and straight and cyclic hydrocarbyl groups. Biomedicines

[0070] ​In some instances, the stereochemistry at each phosphonucleotide bond in the ASO backbone is random. In other instances, the stereochemistry at each phosphonucleotide bond in the ASO backbone is controlled and not random. For example, U.S. Patent No. 9,605,019 describes a method for independently selecting the chiral bias at each phosphorus atom in an oligonucleotide. In some instances, the ASOs used in the compositions and methods provided herein (including, but not limited to, ASOs whose sequences are disclosed herein as SEQ ID NO: 2-351) are ASOs having non-random phosphodiester nucleotide bonds. In some instances, the compositions or compositions used in the methods disclosed herein comprise pure diastereomers of ASO. In other examples, the composition comprises ASO with a diastereomeric 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%.

[0071] In some instances, the ASO has a non-random mixture of Rp and Sp configurations at its phosphonucleotide internucleotide bonds. In some instances, the ASO used in the compositions and methods disclosed herein comprises 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.

[0072] In some instances, the ASO described herein contains a sugar moiety comprising ribose or deoxyribose, or a modified sugar moiety or sugar analogue, including a morpholine ring. Suitable examples of modified sugar moieties include, but are not limited to, 2' substitution, such as 2'- O -Modification, 2'- O -Methyl(2'-) O -Me), 2'- O -Methoxyethyl (2'MOE), 2'- O- aminoethyl, 2'F, N3'->P5' aminophosphoramidate, 2' dimethylaminooxyethoxy, 2' dimethylaminoethoxyethoxy, 2'-guanidinyl, 2'- O - guanidinylethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some examples, the sugar moiety modification is selected from 2'- O - Me, 2'F, and 2' MOE. In other examples, the sugar moiety modification is an additional bridge, such as in a locked nucleic acid (LNA). In some examples, the sugar analog contains a morpholino ring, such as a phosphorodiamidate morpholino (PMO). In some examples, the sugar moiety comprises a ribofuranosyl or 2' deoxyribofuranosyl modification. In some examples, the sugar moiety comprises a 2'4'-constrained 2'- O - methoxyethyl (cMOE) modification. In some examples, the sugar moiety comprises a cEt 2', 4' constrained 2'- O ethyl BNA modification. In other examples, the sugar moiety comprises a tricyclo-DNA (tcDNA) modification. In some examples, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some examples, the sugar moiety comprises a 2'- O - (2-N-methylcarbamoyl ethyl) (MCE). Modifications are known in the art, as exemplified in Jarver et al., 2014, Nucleic Acid Therapeutics (NAT), 24(1): 37-47. Nucleic Acid Therapeutics

[0073] In some examples, each constituent nucleotide of the ASO is modified in the same way, for example, each linkage of the backbone of the ASO comprises a phosphorothioate linkage, or each ribose moiety comprises a 2'- O - methyl modification. In other examples, a combination of different modifications is used, for example, an ASO comprising a combination of phosphorodiamidate linkages and sugar moieties comprising a morpholino ring (morpholino).

[0074] In some examples, the ASO comprises one or more backbone modifications. In some examples, the ASO comprises one or more sugar moiety modifications. In some examples, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some examples, the ASO comprises a 2'- O - MOE modification and a phosphorothioate backbone. In some examples, the ASO comprises a peptide nucleic acid (PNA).

[0075] In some preferred examples, the ASO comprises a phosphorodiamidate morpholino (PMO).

[0076] ​​Those skilled in the art will appreciate that ASOs can be modified in order to achieve a desired property or activity of the ASO or to reduce an undesired property or activity of the ASO. In some examples, the ASO is modified to alter one or more properties. For example, such modifications can: enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequences; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of the ASO into a cell and / or a particular subcellular compartment; alter the pharmacokinetics or pharmacodynamics of the ASO; and / or modulate the half-life of the ASO in vivo.

[0077] In some examples, the ASO comprises one or more 2'-0-methyl (MOE) modified nucleotides, which have been shown to confer significant enhanced resistance of the ASO to nuclease degradation and increased bioavailability. O -(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides, which have been shown to confer significant enhanced resistance of the ASO to nuclease degradation and increased bioavailability.

[0078] Methods for synthesizing and chemically modifying ASOs, as well as synthesizing ASO conjugates, are well known in the art, and such ASOs are commercially available.

[0079] In some examples, the compositions (e.g., pharmaceutical compositions) provided herein include two or more ASOs that are complementary to the same targeted portion of the mRNA 3' UTR but have different chemistries. PKD1 In other examples, two or more ASOs that are complementary to different targeted portions of the mRNA 3' UTR. PKD1 In other examples, two or more ASOs that are complementary to different targeted portions of the mRNA 3' UTR.

[0080] In some examples, the compositions disclosed herein include an ASO linked to a functional moiety. In some examples, the functional moiety is a delivery moiety, a targeting moiety, a detection moiety, a stabilizing moiety, or a therapeutic moiety. In some examples, the functional moiety includes a delivery moiety or a targeting moiety. In some examples, the functional moiety includes a stabilizing moiety. In some preferred examples, the functional moiety is a delivery moiety.

[0081] Suitable delivery moieties include, but are not limited to, lipids, peptides, carbohydrates, polyethers, and antibodies.

[0082] In some examples, the delivery moiety includes a cell-penetrating peptide (CPP).

[0083] Suitable examples of CPPs are described in, for example, PCT / AU2020 / 051397. In some examples, the amino acid sequence of the CPP comprises or consists of: RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 352). In one example, the CPP comprises the sequence RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 352), optionally wherein any amino acid other than glycine is a D amino acid. In other examples, the delivery moiety comprises a receptor binding domain.

[0084] In other examples, the delivery moiety comprises a carbohydrate. In some examples, the carbohydrate delivery moiety is selected from the group consisting of N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc) and mannose. In one example, the carbohydrate delivery moiety is GalNAc.

[0085] In other examples, the delivery moiety comprises a lipid. Examples of suitable lipids as delivery moieties include, but are not limited to, a cholesterol moiety, a cholesteryl group moiety and an aliphatic lipid. In some examples, the delivery moiety comprises a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is about C8 to C20. Examples of suitable fatty acid moieties and conjugation thereof to oligonucleotides are found in, for example, International Patent Publication WO2019232255 and Prakash et al., (2019).

[0086] In further examples, the delivery moiety comprises an antibody, as described in, for example, Dugal-Tessier et al., (2021).

[0087] Suitable examples of stabilising moieties include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) and poly(2-oxazoline)s (POx).

[0088] In some examples, wherein the ASO is linked to a functional moiety, the functional moiety is covalently linked to the ASO. In other examples, the functional moiety is non-covalently linked to the ASO.

[0089] The functional moiety can be linked to one or more of any nucleotides in the ASO at any of several positions on a sugar, base or phosphate group, as understood in the art and described in the literature, for example using a linker. The linker can comprise a bivalent 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.

[0090] In some examples, the composition comprising any one of the ASOs disclosed herein further comprises a delivery nanocarrier complexed with the ASO. In some examples, the delivery nanocarrier is selected from the group consisting of a lipoplex, a liposome, an exosome, an inorganic nanoparticle, and a DNA nanostructure. In other examples, the delivery nanocarrier comprises a lipid nanoparticle encapsulating the ASO. Various delivery ASO-nanocarrier complex forms are known in the art, as reviewed in, for example, Roberts et al., supra.

[0091] Vectors expressing antisense RNA (AR) for PKD1 In some examples, provided herein is a composition containing a vector for expressing an AR in a mammalian cell, the AR being associated with PKD1 3' UTR, wherein the association of the AR with the targeted portion reduces the specific binding of a miRNA to the 3' UTR, for example, the binding of a miR-17 family miRNA (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p) or a miR-200 family miRNA (e.g., miR-200b, miR200c, or miR-429).

[0092] In some examples, the promoter used in the expression vector is a kidney cell type- selective promoter for driving expression of the AR in a mammalian cell. In some examples, the kidney cell type-selective promoter is selective for expression in a kidney cell type selected from the list consisting of: pericyte, podocyte, parietal epithelial cell, proximal tubule cell, ascending limb of the loop of Henle cell, descending limb of the loop of Henle cell, distal tubule cell, connecting tubule cell, intercalated cell, principal cell, peritubular capillary endothelial cell, and glomerular endothelial cell. For example, the sodium-dependent phosphate transporter type 2a (NPT2a) promoter expressed in the proximal tubule, the sodium-potassium-2-chloride co-transporter (NKCC2) promoter expressed in the thick ascending limb of the loop of Henle (TALH), the aquaporin 2 (AQP2) promoter expressed in the collecting duct, and the podocin promoter expressed in the podocyte.

[0093] In some instances, the promoter is an inducible promoter, for example, a ligand-regulated trans-actinant (such as Tet-inducible rtTA), which allows for the titration of AR transcription in target mammalian cells. In some instances, the promoter driving AR expression is a U6 or other Pol III promoter, which is particularly well-suited for transcribing short RNA sequences, such as the AR sequences disclosed herein. In some instances, the expression vector utilizes a hybrid promoter system, for example, a Tet-O-regulated U6 promoter system as described in the following literature: Lin et al. (2004), Letters of the Federation of European Biochemical Societies (FECS). FEBS Letters )》, 577 (2004)376-380. In some instances, where both cell type specificity and inducibility of the AR expression vector are desired, a two-part expression system is used, wherein the expression of a ligand-regulated trans-actor is driven by a cell type-selective promoter, and the expression of AR disclosed herein is driven by a ligand-regulated trans-actor-regulated promoter.

[0094] In some instances, the expression vectors used in the compositions disclosed herein are non-viral expression vectors, such as plasmid vectors, small circular DNA vectors, linear amplicon expression cassettes, etc.

[0095] In some instances, compositions containing non-viral expressed viruses further comprise a transfection agent. Exemplary transfection agents for transfection include, but are not limited to, jet-PEI. ® (Available from Polyplus-transfection in Strasbourg, France) ® SA Company (Polyplus-transfection) ® (SA, Strasbourg, France) obtained); TurboFect in vivo transfection reagent (Thermo Fisher Scientific) and cationic derivatives of polyisoprene alcohol (PTAI), such as Rak et al. (2016), Journal of Experimental Medicine ( J Gene Med As described in 》, 18(11-12):331-342.

[0096] In other instances, the expression vector to be used is a viral vector, that is, a non-replicating recombinant virus suitable for expressing the AR disclosed herein.

[0097] Preferably, used to express PKD1Recombinant viruses of AR are DNA viruses. Suitable types of DNA viruses include adeno-associated virus (AAV), adenovirus, lentivirus, herpes simplex virus (HSV), and ring virus. Methods for designing, producing, and using this type of recombinant DNA virus have been established in the art, as illustrated in the following literature: Fukazawa et al., (2010), *International Journal of Molecular Medicine* (…). International J of Mol. Med )》, 25(1), 3-10, and “gene therapy regimens” for adenovirus; “Adeno-associated virus: methods and protocols” for AAV; Cody et al. (2013), Journal of Genetic Syndromes and Gene Therapy ( Journal of Genetic Syndromes & Gene Therapy )》, 4(1),126, and “Herpes simplex virus: methods and protocols” for HSV; “Gene therapy protocols, Volume 1: generation and in vivo application of gene transfer vectors”; and Merten et al. (2016), “Molecular therapy: methods and clinical developments”. Molecular Therapy - Methods & Clinical Development ) , 3, 16017; and Emeagi et al. (2013) on lentiviruses, Current Molecular Medicine ( Current Molecular Medicine )》 13(4), 602-625. In some preferred examples, the viral vector is recombinant AAV.

[0098] Genetically modified cells This document also provides information on genetically modified cells. In some instances, the genetically modified cells are genetically modified bacterial cells (e.g., recombinant *E. coli* used to amplify the AR expression vectors disclosed herein). In other instances, the genetically modified cells are genetically modified mammalian cells that have been transfected with any of the ASO or non-viral AR expression vectors, or transduced with any of the viral AR expression vectors disclosed herein. In some instances, the genetically modified mammalian cells are in vitro, for example, as a cultured cell population. In other instances, the genetically modified mammalian cells are in vivo, for example, in mice. In some instances, the genetically modified mammalian cells are human cells.

[0099] In some examples, the genetically modified mammalian cell is a primary cell type. Suitable examples of primary cell types include, but are not limited to, cyst cells, pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending loop of Henle cells, descending loop of Henle cells, distal tubular cells, juxtaglomerular cells, interstitial cells, principal cells, peritubular capillary endothelial cells, and glomerular endothelial cells. In some examples, such primary cell types can be obtained by differentiation of a human pluripotent stem cell line (e.g., a human induced pluripotent stem cell (hiPSC) line or a human embryonic stem cell (hESC) line). Methods for obtaining a variety of different kidney cell types are known in the art, as reviewed in, e.g., de Carvalho Ribeiro et al. (2020) and Osafune et al. (2021). In some examples, the primary cell type is derived from divided kidney tissue, such as a kidney cyst. Cyst cells excised from ADPKD kidneys have been widely used since the 1980s. A detailed protocol for cyst formation in vitro was recently published by Sharma et al. (2019). In other examples, the genetically modified mammalian cell is derived from a cell line. In some examples, the cell line is a pluripotent stem cell line (e.g., a hiPSC or hESC) or a human kidney cell line. In some examples, the genetically modified mammalian cell is derived from a HEK293, HK-2, or WT9-7 cell line. In some preferred examples, the genetically modified mammalian cell endogenously expresses polycystin 1. In some embodiments, genetically modified human cells are provided in kidney organoids, e.g., by differentiating human pluripotent stem cells or human adult stem cell-derived kidney organoids as reviewed in, e.g., Kang (2023), Development and Reproduction (Dev Reprod), 27(2):57-65. Development & Reproduction In some examples, the genetically modified mammalian cell is a primary cell type. Suitable examples of primary cell types include, but are not limited to, cyst cells, pericytes, podocytes, parietal epithelial cells, proximal tubular cells, ascending loop of Henle cells, descending loop of Henle cells, distal tubular cells, juxtaglomerular cells, interstitial cells, principal cells, peritubular capillary endothelial cells, and glomerular endothelial cells. In some examples, such primary cell types can be obtained by differentiation of a human pluripotent stem cell line (e.g., a human induced pluripotent stem cell (hiPSC) line or a human embryonic stem cell (hESC) line). Methods for obtaining a variety of different kidney cell types are known in the art, as reviewed in, e.g., de Carvalho Ribeiro et al. (2020) and Osafune et al. (2021). In some examples, the primary cell type is derived from divided kidney tissue, such as a kidney cyst. Cyst cells excised from ADPKD kidneys have been widely used since the 1980s. A detailed protocol for cyst formation in vitro was recently published by Sharma et al. (2019). In other examples, the genetically modified mammalian cell is derived from a cell line. In some examples, the cell line is a pluripotent stem cell line (e.g., a hiPSC or hESC) or a human kidney cell line. In some examples, the genetically modified mammalian cell is derived from a HEK293, HK-2, or WT9-7 cell line. In some preferred examples, the genetically modified mammalian cell endogenously expresses polycystin 1. In some embodiments, genetically modified human cells are provided in kidney organoids, e.g., by differentiating human pluripotent stem cells or human adult stem cell-derived kidney organoids as reviewed in, e.g., Kang (2023), Development and Reproduction (Dev Reprod), 27(2):57-65.

[0100] The genetically modified cells disclosed herein can be genetically modified by any of a number of methods and strategies known in the art (e.g., transient transfection, stable transfection, and viral transduction). In some examples, transfection with an ASO or non-viral vector is performed by nucleofection. In other examples, cell transfection is performed by lipofection.

[0101] Pharmaceutical compositions Also provided herein are pharmaceutical compositions comprising any of the foregoing ASOs, non-viral expression vectors, modified messenger RNAs (mmRNAs), and viral expression vectors disclosed herein, and formulated with at least one pharmaceutically acceptable excipient, including a carrier, a filler, a preservative, an adjuvant, a solubilizer, and / or a diluent.

[0102] Pharmaceutical compositions containing any of the ASOs or expression vector compositions described herein for use in the methods disclosed herein can be prepared according to conventional techniques known in the pharmaceutical industry and described in the disclosed literature. In some examples, a pharmaceutical composition for treating a subject comprises a therapeutically effective amount of any of the ASOs or expression vectors disclosed herein.

[0103] Pharmaceutically acceptable salts are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, 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, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0104] In some examples, the pharmaceutical compositions are formulated into any of a variety of possible dosage forms, including, but not limited to, topical ointments, intravenous administration solutions, subcutaneous injections, intrathecal administration, intracistema magna administration, tablets, capsules, gel capsules, liquid syrups, and soft gels. In some examples, the compositions are formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers. In some examples, the pharmaceutical formulations disclosed herein are provided in a form including, but not limited to, solutions, emulsions, microemulsions, foams, or formulations containing liposomes (e.g., cationic or non-cationic liposomes).

[0105] In some examples, a pharmaceutical formulation comprising any of the ASOs or expression vectors described herein can include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients appropriate and known to the skilled artisan. In some examples, where the pharmaceutical composition includes a liposome, such liposomes can also include sterically stabilized liposomes, for example, liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes. In some examples, sterically stabilized liposomes comprise one or more glycolipids or are derivatized with one or more hydrophilic polymers, such as a PEG moiety. In some examples, a surfactant is included in the pharmaceutical formulation.

[0106] In some examples, the pharmaceutical composition further includes a penetration enhancer to enhance delivery of the ASO or non-viral expression vector, for example, to aid in diffusion across a cell membrane and / or to enhance the permeability of a lipophilic drug. In some examples, the penetration enhancer includes a surfactant, a fatty acid, a bile salt, or a chelating agent.

[0107] In some examples, the pharmaceutical composition comprises an ASO or non-viral vector at a dose ranging from about 0.0001 mg / kg to about 80 mg / kg (e.g., 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 ranging from about 0.01 mg / kg to about 80 mg / kg.

[0108] In some examples, the pharmaceutical composition comprises a plurality of ASOs or AR expression vectors. In some examples, the pharmaceutical composition comprises another drug or therapeutic agent suitable for treating a subject having a condition associated with inflammation in addition to the ASO or AR expression vector.

[0109] Methods As described herein, ADPKD is associated with insufficient levels of functional polycystin 1. Accordingly, the methods described herein include methods of treating ADPKD by administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising any of the ASOs or expression vectors disclosed herein. Likewise, in some examples, any of the ASOs or AR expression vectors disclosed herein are used in the preparation of a medicament for reducing inflammation.

[0110] Also provided herein is a method for increasing the level of PKD1 mRNA and subsequently polycystin 1 protein in an ex vivo cell or in vivo tissue, the method comprising contacting the cell with an ASO, AR expression vector, or pharmaceutical composition as disclosed herein, whereby cell abnormalities associated with ADPKD, such as cyst formation, are reduced.

[0111] In some embodiments, administration of any of the ASOs, AR expression vectors, or pharmaceutical compositions disclosed herein to a subject or contacting of a cell with any of the ASOs, AR expression vectors, or pharmaceutical compositions disclosed herein increases the level of PKD1 mRNA or polycystin 1 protein by about 1.1-fold to about 3-fold, such as 1.3-fold to about 2.7-fold, about 1.4-fold to about 2.6-fold, about 1.5-fold to about 2.5-fold, about 1.6-fold to about 2.4-fold, about 1.7-fold to about 2.3-fold, about 1.8-fold to about 2.2-fold, or PKD1 mRNA or polycystin 1 protein by about 1.1-fold to about 3-fold.

[0112] Suitable routes of administration for treatment with the compositions, pharmaceutical compositions, or medicaments disclosed herein include, but are not limited to, intravenous, intra-arterial, subcutaneous, intrathecal, oral, and topical administration.

[0113] In some examples, any of the treatment methods disclosed herein can optionally include the step of determining the level of PKD1 mRNA, polycystin 1 protein in the subject prior to and / or after treatment.

[0114] As will be appreciated by those skilled in the art, the treatment methods disclosed herein include administration of the compositions and pharmaceutical compositions disclosed herein to a subject (e.g., a human subject) in a therapeutically effective amount. As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of a disclosed ASO, non-viral or viral expression vector administered sufficient to relieve to some extent one or more symptoms and / or clinical indicia associated with pathologic inflammation in a particular disease or health condition. In some examples, an “effective amount” for therapeutic use is the amount of one of the foregoing agents that is required to provide a clinically significant reduction of disease symptoms and / or inflammatory markers, or to prevent disease symptoms, without undue adverse side effects. The appropriate “effective amount” in any individual case can be determined using techniques, such as a dose escalation study. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. It will be appreciated that the “effective amount” or “therapeutically effective amount” can vary from subject to subject, due to variation in the age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician of the subject. By way of example only, therapeutically effective amounts can be determined by routine experimentation in light of the disclosure, including but not limited to dose escalation clinical trials. When more than one therapeutic agent is used in combination, the “therapeutically effective amount” of each therapeutic agent can refer to the amount of the therapeutic agent that is therapeutically effective when used alone, or can refer to a reduced amount that is therapeutically effective due to use in combination with one or more additional therapeutic agents.

[0115] Combination therapy Pharmaceutical compositions comprising any of the ASOs or AR expression vectors disclosed herein can also be used in combination with other agents of therapeutic value for treating conditions associated with pathologic inflammation. Generally, the other agents need not necessarily be administered in the same pharmaceutical composition, and due to different physical and chemical properties, can preferably be administered by different routes. Where possible, determination of the administration mode and administration rationality in the same pharmaceutical composition is well within the knowledge of the skilled clinician. Initial administration can be made according to established protocols known in the art, and then based on the observed effects, the skilled clinician can modify the dosage, administration mode, and administration time.

[0116] Depending on the stage and progression of the inflammatory disease to be treated, the patient’s condition, and the selection of the particular therapeutic agents used, the compositions and pharmaceutical compositions comprising the ASOs and / or expression vectors and additional therapeutic agents can be administered concurrently (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen) or sequentially. Determination of the order of administration and the number of repetitions of administration of each therapeutic agent during the treatment regimen is well within the knowledge of the skilled clinician after evaluating the disease being treated and the patient’s condition.

[0117] It is known to those skilled in the art that the therapeutically effective dose can vary when drugs are used in a therapeutic combination. Methods for experimentally determining the therapeutically effective dose of drugs and other agents for a combination treatment regimen are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been extensively described in the literature. Combination therapy further includes periodic treatment that is initiated and stopped at different times to aid in the clinical management of the patient.

[0118] For combination therapy, the dosage of the co-administered therapeutic will, of course, vary depending on the type of adjunctive agent, ASO or expression vector employed, and the stage of the patient's disease to be treated.

[0119] Pharmaceutical compositions comprising the ASO, AR or expression vector making up the combination therapy disclosed herein and the additional therapeutic agent can be in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical compositions making up the combination therapy can also be administered sequentially, where either therapeutic is administered by a regimen requiring two-step administration. Two-step administration regimens can entail sequential administration of the active agents or spaced-apart administration of the separate active agents. Depending on the properties of each agent, such as potency, solubility, bioavailability, plasma half-life, and kinetic characteristics of the agent, the time period between the multiple administration steps can range from minutes to hours. Diurnal variations in various physiological parameters can also be evaluated to determine the optimal dose interval.

[0120] Examples of suitable therapeutic agents co-administered 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-2 receptor blockers, paracetamol, opioid drugs, and antibiotics.

[0121] Examples Example 1: PKD1 Identification and sequence selection of 3' UTR target sequences.

[0122] PKD1 Mutations in PKD1 and PKD2 lead to misregulation and / or insufficient levels of functional polycystin-1 associated with the onset and severity of ADPKD. miRNAs from 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 the mRNA, leading to increased polycystin-1 protein. PKD1

[0123] ​For the 3' UTR upregulation strategy, PMO (SEQ ID NO: 2-351 or 353-362) is designed as part of the microwalking strategy to span the entire PKD1 The 3' UTR (SEQ ID NO: 1) thus encompasses 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 having sequences corresponding to SEQ ID NO: 47, 334, and 335 are complementary to the binding sites of miR-17 family miRNAs (Fig. 1B), and PMOs having sequences corresponding to SEQ ID NO: 14, 73, 75, 336, and 337 are complementary to the binding sites of miR-200 family miRNAs (Fig. 1C). The miRNA binding sites are highlighted in bold (Figs. 1B and 1C).

[0124] Example 2: Screening for targets in HEK293 cells PKD1 The miR-17 binding site in the middle can enhance PKD1 PPMO expressed by transcripts.

[0125] 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) were conjugated with a cell-penetrating peptide (SEQ ID NO: 352) to generate peptide-PMO (PPMO). HEK293 cells were treated with PPMO or a control for 24 hours, and total RNA was extracted using the MagMAX Total RNA 96 Extraction Kit. The RNA was evaluated by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 Gene expression. PKD1 Transcript expression relative to housekeeping TATA-binding protein ( TBP TaqMan (probe catalog number Hs00427620_m1) was normalized, and the changes were calculated as folds of change compared to untreated cells.

[0126] As shown in Figure 2, compared with untreated cells, PPMO corresponding to SEQ ID NO: 47 (+604+628), 334 (+611+635), and 335 (+615+639) induced [the cells to be treated]. PKD1Dose-dependent increase of 1.1-fold or more in mRNA. Non-targeting control (GTC CTR) predicted not to hybridize to human transcripts was included as a sham treatment.

[0127] Example 3: Effect of PPMO on PC1 protein levels in ADPKD patient cell lines masks sequence in 3’ UTR of PKD1 targeted by miR-17 miRNA seed sequence According to Example 2, PPMOs showing significant PKD1 PPMOs capable of upregulating polycystin 1 (PC1) protein in ADPKD patient cell lines mRNA upregulation. ADPKD patient cell lines were established by immortalizing cells from a single proximal cortical tubular cyst taken from a patient with ADPKD carrying a heterozygous mutation in PKD1 (p.Q2556*). PPMOs were dissolved in molecular grade H2O to create a 1 mM stock solution. The stock solution was diluted in culture medium and administered at 10 µM with n = 3 technical replicates per treatment condition. Untreated cells were treated with an equal volume of culture medium only. Cells were incubated for 5 days before measuring the presence of polycystin 1 (PC1) on the cell surface by flow cytometry. Cells were incubated with an anti-rabbit polycystin 1 antibody (ab74115, Abeam) followed by a goat anti-mouse antibody conjugated with Alexa Fluor 488 (A-11001, Thermo Fisher). As demonstrated in Figure 3, the median fluorescence intensity (MFI) of each sample was compared to untreated cells (UT) to identify PMOs that increased expression of polycystin 1. PPMOs corresponding to SEQ ID NOs: 47 (+604+628), 334 (+611+635), and 335 (+615+639) induced a 1.1-fold or more increase in polycystin 1 protein compared to untreated cells, respectively.

[0128] Example 4: Functional validation of PPMOs in patient-derived primary cell 3D cyst model.

[0129] PPMOs showing significant polycystin 1 protein upregulation were selected for functional validation in patient-derived 3D cyst models according to Example 3. The models utilize cyst cells extracted from kidneys donated by ADPKD patients. When grown ex vivo, these cells spontaneously form cysts in 3D matrices and can be used to assess the functional effects of drug treatments on cyst growth. Immediately after seeding, patient cells were co-exposed to treatments. PPMOs were dissolved in molecular grade H2O and administered at 1 µM, 3 µM, 10 µM, and 20 µM, with n = 4 technical replicates per treatment condition. Control cells were treated with an equal volume of H2O only. After 7 days of exposure, cultures were fixed and stained for actin cytoskeleton and nuclei. Growth and distension of cysts were visualized by high-content microscopy imaging and images were analyzed using Ominer® image analysis software. FIG. 4 shows representative images of 3D patient cyst size after treatment with 20 µM PPMO with sequences corresponding to SEQ ID NO: 47 (+604+628), 334 (+611+635), and 335 (+615+639).

[0130] Example 5: Quantification of patient-derived 3D cyst area and cell death to determine therapeutic index.

[0131] All images from the assay described in Example 4 were further analyzed to determine the dose-dependent changes in cyst area (µm2) and cell death (%) for PPMOs with sequences corresponding to SEQ ID NO: 47 (+604+628), 334 (+611+635), and 335 (+615+639). See FIG. 5. 2

[0132] The PPMO corresponding to SEQ ID NO: 47 (+604+628) showed efficacy in inhibiting cyst growth in a dose-dependent manner with no evidence of cytotoxicity at up to 20 µM (FIGS. 5A and 5D). The PPMO corresponding to SEQ ID NO: 334 (+611+635) potently inhibited cyst growth, however slight cytotoxicity was observed at 20 µM treatment (FIGS. 5B and 5E). The PPMO corresponding to SEQ ID NO: 335 (+615+639) was found to be cytotoxic at 20 µM. However, the PPMO showed moderate inhibition of cyst growth at 10 µM treatment in the cyst model derived from ADPK patients (FIGS. 5C and 5F).

[0133] Example 6: Optimization of PMO sequence to enhance PKD1 upregulation by inhibiting miR-17 binding to the 3’ UTR of the PKD1 transcript ​PMOs (SEQ ID NOS: 353-361) were designed to have some modifications to micro-walk SEQ ID NO: 47, such as length shortening and engineered base mismatches. The micro-walk PMOs were conjugated to a cell penetrating peptide (SEQ ID NO: 352) to create PPMOs and tested for their efficacy in PKD1 upregulation in HEK293 cells. HEK293 cells were treated with PPMOs or controls for 24 hours. Subsequently, total RNA was extracted using the MagMAX Total RNA 96 Extraction Kit method. PKD1 transcript expression was assessed by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 transcript expression was normalized to the housekeeping TATA-binding protein (TBP, TaqMan, probe catalog number Hs00427620_m1) and DexH-box helicase 57 (DHX57, Thermo Fisher Scientific; assay ID Hs00376574_m1), and changes were calculated as fold change compared to untreated cells. The results in FIG. 6 show that PKD1 was slightly upregulated (1.1 fold) when treated with PPMO (+604+626) MM16, (+604+628) MM16, (+604+626), (+604+623), (+604+628), and (+604+627).

[0134] Example 7: Screening PPMOs that enhance PKD1 upregulation by inhibiting miR-200 binding to the 3’ UTR of the PKD1 transcript PMOs having sequences corresponding to SEQ ID NOS: 14, 73, 75, 336-337 were designed to target binding to the 3’ UTR of the PKD1 mRNA to prevent binding of miR-200. The designed PMOs were conjugated to a cell penetrating peptide (SEQ ID NO: 352) to create peptide-PMOs (PPMOs) and tested for their efficacy in HEK293 cells. After 24 hours of PPMO treatment, total RNA was extracted using the MagMAX Total RNA 96 Extraction Kit method. PKD1 gene expression was assessed by digital droplet PCR (TaqMan; probe catalog number Hs00947394_g1). PKD1 transcript expression was normalized to the housekeeping TATA-binding protein (TBP, TaqMan, probe catalog number Hs00427620_m1), and changes were calculated as fold change compared to untreated cells. As shown in FIG. 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 (1.1 fold) compared to untreated cells.

[0135] Example 8: PC1 upregulation evaluation of PPMO-mediated inhibition of miR-17 binding in HEK293 cells PPMOs with sequences corresponding to SEQ ID NO: 47 (+604+628) and 334 (+611+635) were incubated with HEK293 for 5 days. The level of PC1 upregulation was evaluated using Western blot assay. Proteins were extracted using RIPA buffer supplemented with 2% protease inhibitor cocktail and 2x PhosSTOP at day 5 post-treatment. Protein lysates were cleared and total proteins were quantified using BCA protein kit. Samples were run on NuPAGE 3-8% Tris-Acetate protein gel. Proteins were transferred to nitrocellulose membrane by wet transfer. Membranes were stained for total protein and mouse anti-PC1 (Santa-Cruz, Cat# sc130554) primary antibody, followed by anti-mouse (pre-adsorbed IRDye ® 800CW) secondary antibody. Blots were imaged on Odyssey Imager and quantification analysis was performed using ImageStudio Ver 5.5 software (as shown in Figure 8). Raw fluorescence signal of PC1 was first normalized to that of loading control (total protein) and then expressed as 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 up to 1.64-fold at day 5 compared to UT. Non-targeting control, which is not predicted to hybridize to human transcripts, was included as a negative control.

[0136] Example 9: Time course and dose range analysis of PPMO-induced PC1 protein in cell lines derived from ADPKD patients.

[0137] Cell lines derived from ADPKD patients were incubated with PPMO targeting PC1 (SEQ ID NO: 47) and PPMO targeting miR-17 (SEQ ID NO: 334) for 5 days. The level of PC1 upregulation was evaluated using Western blot assay. Proteins were extracted using RIPA buffer supplemented with 2% protease inhibitor cocktail and 2x PhosSTOP at day 5 post-treatment. Protein lysates were cleared and total proteins were quantified using BCA protein kit. Samples were run on NuPAGE 3-8% Tris-Acetate protein gel. Proteins were transferred to nitrocellulose membrane by wet transfer. Membranes were stained for total protein and mouse anti-PC1 (Santa-Cruz, Cat# sc130554) primary antibody, followed by anti-mouse (pre-adsorbed IRDye PKD1WT9-7 cell line with nonsense mutation (p.Q2556*) was incubated with PPMO corresponding to SEQ ID NO: 47 (+604+628) at concentrations of 30 µM, 60 µM, 90 µM and 120 µM for 2 days, 3 days and 5 days. PC1 protein levels were assessed to evaluate the efficacy of PPMO. Protein extraction was performed using RIPA buffer supplemented with 2% protease inhibitor cocktail and 2x PhosSTOP. Protein lysates were cleared and total protein was quantified using BCA protein kit. Samples were run on NuPAGE 3-8% Tris-acetate protein gels. Proteins were transferred to nitrocellulose membranes by wet transfer. Membranes were stained for total protein and mouse anti-PC1 (Santa Cruz, Cat# sc130554) primary antibody and then anti-mouse (pre-adsorbed IRDye® 800CW) secondary antibody. Blots were imaged on an Odyssey Imager and quantified using ImageStudio Ver 5.5 software. Raw fluorescence signal for PC1 was first normalized to that of the loading control (total protein) and then expressed as fold change relative to UT. In Figure 10, the western blot images show PC1 expression. PPMO (+604+628) - SEQ ID NO: 47 induced a dose-dependent increase in PC1 protein levels, up to 1.36-fold of UT. Maximum PC1 upregulation was observed at day 2 and plateaued at 90 µM.

[0138] Those skilled in the art will appreciate that numerous changes and / or modifications can be made to the present application as shown in the particular examples without departing from the spirit or scope of the application as broadly described. The present application examples are, therefore, to be considered in all respects as illustrative and not restrictive.

[0139] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0140] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present application. It is not to be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present application.

[0141] References de Carvalho Ribeiro et al., 2020, Stem Cells International, doi.org / 10.1155 / 2020 / 8894590.

[0142] Dugal-Tessier et al., (2021), Journal of Clinical Medicine, 10(4):838. J Clin Med.

[0143] Osafune et al., 2021, Clinical and Experimental Nephrology, 25(6):574-584.

[0144] Prakash et al., 2019, Nucleic Acids Research, 47(12):6029-6044.

[0145] Sharma et al., 2019, Methods Cell Biol., doi.org / 10.1016 / bs.mcb.2019.05.008 Appendix 1: Sequences and SEQ ID NOs SEQ ID NO: 1 Human PKD1 Transcript 3' UTR (PKD-201 canonical transcript ENST00000262304.9) ​ Table 1: Targeting PKD1 Exemplary ASO or AR sequences for 3' UTR sequences SEQ ID NO: 352. Amino acid sequence of CPP RRSRTARAGRPGRNSSRPSAPR

Claims

1. An antisense oligonucleotide associated with polycystic kidney disease 1 ( PKD1 The binding of the targeted portion of the 3' untranslated region (UTR) of the mRNA, wherein the binding of the antisense oligonucleotide to the targeted portion increases the level of polycystin 1 protein.

2. An antisense oligonucleotide, which is associated with polycystic kidney disease 1 ( PKD1 The antisense oligonucleotide binds to the targeted portion of the 3' untranslated region (UTR) of the mRNA, wherein when introduced into a 3D kidney cyst culture, the binding of the antisense oligonucleotide to the targeted portion reduces kidney cyst growth or decreases kidney cyst size.

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

4. The antisense oligonucleotide of any one of claims 1-3, wherein the antisense oligonucleotide comprises the sequence of any one of SEQ ID NOs: 2-351 or 353-362.

5. A vector for expressing antisense RNA (AR) in mammalian cells, said AR being associated with polycystic kidney disease 1 (… PKD1 The AR binds to the targeted portion of the 3' UTR of the mRNA, wherein when introduced into a 3D kidney cyst culture, the binding of the AR to the targeted portion reduces cyst growth.

6. A vector for expressing an antisense RNA (AR) in a mammalian cell, the AR binding to a targeted portion of the 3' UTR of polycystic kidney disease 1 (PKD1) mRNA, wherein binding of the AR to the targeted portion increases the level of polycystin 1 protein. PKD1 ) 7. The vector of claim 5 or 6, wherein binding of the AR to the targeted portion reduces specific binding of a miR-17 family member (e.g., miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5) or a miR-200 family member (e.g., miR-200b, miR200c, or miR-429) to the 3' UTR.

8. The vector of any one of claims 5-7, wherein the AR comprises the sequence of any one of SEQ ID NOs: 2-351 or 353-362.

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

10. The vector of claim 9, wherein the kidney cell type selective promoter is selective for expression in a kidney cell selected from the list consisting of: pericyte, podocyte, parietal epithelial cell, proximal tubule cell, ascending loop of Henle cell, descending loop of Henle cell, distal tubule cell, connecting tubule cell, intercalated cell, principal cell, peritubular capillary endothelial cell, and glomerular endothelial cell.

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

12. The vector of any one of claims 6-11, wherein the vector is a non-viral vector.

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

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

15. The antisense oligonucleotide of any one of claims 1-3 or the vector of any one of claims 6, 7, or 9-14, wherein the antisense oligonucleotide or the antisense RNA binds within the targeted portion of the 3' UTR corresponding to SEQ ID NO:

1.

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

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

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

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

20. The antisense oligonucleotide or vector of claim 19, wherein the nucleotide sequence comprises or consists of any one of SEQ ID NOs: 334 or 335.

21. The antisense oligonucleotide or vector of claim 19, wherein the nucleotide sequence comprises or consists of any one of SEQ ID NOs: 355-360.

22. The antisense oligonucleotide or vector of 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 of claim 19, wherein the nucleotide sequence comprises or consists of SEQ ID NO:

47.

24. The antisense oligonucleotide of any one of claims 1-3 or 15-23, wherein the antisense oligonucleotide comprises a backbone modification.

25. The antisense oligonucleotide of claim 24, wherein the backbone modification comprises a phosphorothioate bond or a phosphorodiamidate bond.

26. The antisense oligonucleotide of claim 24 or claim 25, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino, a locked nucleic acid, a 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 of any one of claims 24-26, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

28. The antisense oligonucleotide of claim 27, wherein each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

29. The antisense oligonucleotide of any one of claims 24-26, wherein the antisense oligonucleotide comprises 2’- modified O -methoxyethyl moieties.

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

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

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

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

34. The antisense oligonucleotide of any one of claims 1-4 or 15-33, wherein the antisense oligonucleotide further comprises a linked functional moiety.

35. The antisense oligonucleotide of claim 34, wherein the functional moiety comprises a delivery moiety.

36. The antisense oligonucleotide of claim 35, wherein the delivery moiety is selected from the group consisting of a lipid, a peptide, a polyether, a carbohydrate, and an antibody.

37. The antisense oligonucleotide of claim 35 or claim 36, wherein the delivery moiety comprises a cell penetrating peptide (CPP).

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

352.

39. The antisense oligonucleotide of claim 35 or claim 36, wherein the delivery moiety comprises a receptor binding domain (RBD).

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

41. The antisense oligonucleotide of claim 34, wherein the functional moiety comprises a stabilizing moiety.

42. The antisense oligonucleotide of any one of claims 34-41, wherein the functional moiety is covalently linked to the antisense oligonucleotide.

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

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

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

46. A pharmaceutical composition comprising the antisense oligonucleotide or vector of any one of claims 1-45, and a pharmaceutically acceptable excipient.

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

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

49. The method of claim 47 or claim 48, wherein kidney cyst growth is reduced or kidney cyst size is reduced.

50. Use of the antisense oligonucleotide or vector of any one of claims 1-45 for the manufacture of a medicament for the treatment of ADPKD.

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

52. The method of claim 51, wherein the cell is in a 3D human kidney cyst culture.

53. A genetically modified cell comprising the antisense oligonucleotide or vector of any one of claims 1 to 41.

54. The genetically modified cell of claim 53, wherein the genetically modified cell is a mammalian cell.

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

Images