Compositions and methods for treatment of kidney disease

Abstract

The present disclosure provides a method and related compositions for treating autosomal dominant polycystic kidney disease (ADPKD) comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an antisense oligonucleotide that binds to a targeted portion of the 3' untranslated region (UTR) of Polycystic Kidney Disease 1 (PKD1) mRNA and reduces specific binding of a miR-17 family member to the 3' UTR and a cell penetrating peptide (CPP).

Description

[0001] COMPOSITIONS AND METHODS FOR TREATMENT OF KIDNEY DISEASE

[0002] Related Application Data

[0003] This application claims priority to Australian Provisional Patent Application 2024903650 filed on 8 November 2024 and Australian Provisional Patent Application 2025903153 filed on 22 July 2025, the contents of which are incorporated by reference in their entirety herein.

[0004] Field

[0005] The present disclosure generally relates to methods for treating conditions associated with mutations in the Polycystic Kidney Disease 1 (PKD1) gene.

[0006] Background

[0007] Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited nephropathy that leads to end-stage renal disease (ESRD). Approximately 1 in 500-2,500 individuals carry a mutation for this condition. ADPKD is a progressive disorder that is characterised by an abnormal expansion of renal tubule cells resulting in the growth of multiple cysts in the kidney.

[0008] Cyst development starts early in life and in the more severe cases in utero and is commonly followed by a prolonged period of asymptomatic progression. Signs and symptoms of ADPKD usually present between the ages of 30 and 40. However, approximately 3% of ADPKD patients have either very-early-onset or unusually rapid progressive disease. Consistent with the decline in renal function patients present with various urinary complications such as cyst and urinary' tract infections, a decline in glomerular filtration rate, chronic lower back pain and hypertension. ADPKD patients often present with additional extra-renal conditions including hepatic cysts (in >90% of patients aged >35 years), pancreatic cysts, intracranial aneury sms, colon diverticulosis, and heart valve defects. Overall, ADPKD is associated with significant morbidity a reduced life expectancy.

[0009] ADPKD is predominantly caused by a mutation in one of the poly cystin genes; PKD1 (Poly cystin 1, Transient Receptor Potential Channel Interacting) (74-85% of patients) or PKD2 (15-26%), which results in haploinsufficiency of the proteins encoded by the respective genes. The phenotype of patients with a PKD1 mutation is usually more severe compared to those with PKD2 mutations, which is reflected in approximately' 20 years difference in the mean age of ESRD (54.3 years in PKD1 vs 74.0 years in PKD2 disease).

[0010] There is an ongoing need to provide new treatments or preventative measures for ADPKD. Summary

[0011] In work leading up to the present disclosure, the inventors found that they could systemically, e.g., intravenously, administer a compound conjugated to a cell penetrating peptide (CPP) described herein and the compounds would be preferentially delivered to the kidney and, optionally, the liver. The inventors additionally found that the compound was delivered to cyst forming cells or cyst lining cells in an animal model of ADPKD.

[0012] As exemplified herein, the inventors conjugated an antisense oligonucleotide (ASO), that binds to a targeted portion of the 3' untranslated region (UTR) of a PKD1 mRNA and reduces specific binding of a miR-17 family member to the 3' UTR, to a CPP and systemically delivered the CPP-ASO conjugate to an accepted mouse model of ADPKD. The inventors demonstrated delivery7of the CPP-ASO to various cells within the kidney, including cyst forming cells or cyst lining cells. In cells with a haploinsufficient background (z.e., one mutated allele and one wildtype allele), masking of the miR-17 binding site in the 3' UTR of a wildtype allele PKD1 mRNA results in increased net levels of wildtype PKD1 mRNA and increased Polycystin 1 protein levels.

[0013] Additionally, the inventors showed they could deliver a CPP-ASO conjugate to the kidney of non-human primates. Based on these studies, the inventors determined a dose effective for delivery of a conjugate of the disclosure to the kidney of a human, e.g., to treat ADPKD is 0.4-5.0 mg / kg. e.g., 0.4-1.2 mg / kg or 1.2-2.4 mg / kg. For example, the inventors determined a dose effective for delivery of a conjugate of the disclosure to the kidney of a human, e.g., to treat ADPKD is 0.3 -5.0 mg / kg, e.g., 1.0-2.3 mg / kg.

[0014] Based on the data disclosed herein, the present disclosure provides a method for delivering a compound to a kidney of a subject, the method comprising systemically administering a conjugate comprising a CPP comprising or consisting of SEQ ID NO: 13 linked to or conjugated to the compound.

[0015] In one example, the conjugate is administered parenterally. For example, the conjugate is administered intravenously.

[0016] In one example, the conjugate is preferentially detected in the kidney at least 2 days or 28 days following administration. For example, about 4 times or 5 times or 6 times more conjugate is detected in the kidney than the liver at least 2 days following administration.

[0017] In one example, the compound is delivered to the cortex or the medulla of the kidney. In one example, the compound is delivered to a greater degree to the cortex than the medulla of the kidney.

[0018] In one example, the compound is delivered to a cyst forming cell or a cyst lining cell in the kidney.

[0019] In one example, the compound is an ASO.

[0020] The present disclosure also provides a method for treating ADPKD. the method comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an ASO that binds to a targeted portion of the 3' untranslated region (UTR) of a Polycystic Kidney Disease 1 (PKD1) mRNA and reduces specific binding of a miR-17 family member to the 3' UTR and a CPP comprising the sequence set forth in SEQ ID NO: 13. In one example, the ADPKD is caused by a mutation in a single Poly cystin 1, Transient Receptor Potential Channel Interacting (PKDl) allele in a subject resulting in Polycystin 1 (PCI) haploinsufficiency.

[0021] The disclosure also provides a method for treating autosomal dominant polycystic kidney disease (ADPKD) caused by a mutation in a single Poly cystin 1, Transient Receptor Potential Channel Interacting (PKDP) allele in a subject resulting in Polycystin 1 (PCI) haploinsufficiency, the method comprising systemically administering to the subject a conjugate comprising an antisense oligonucleotide (ASO) that binds to a targeted portion of the 3' untranslated region (UTR) of a wildtype PKD1 allele mRNA and reduces specific binding of a miR-17 family member to the 3' UTR and a cell penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 13.

[0022] The disclosure additionally provides a method for treating ADPKD, the method comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an ASO that binds to a targeted portion of the 3' untranslated region (UTR) of a Polycystic Kidney Disease 1 (PKD1) mRNA and reduces specific binding of a miR-17 family member to the 3' UTR and a CPP comprising the sequence set forth in SEQ ID NO: 13. In one example, the ADPKD is caused by a mutation in a single Poly cystin 1, Transient Receptor Potential Channel Interacting (PKDP) allele in a subject resulting in Polycystin 1 (PCI) haploinsufficiency.

[0023] The disclosure additionally provides a method for increasing expression of PKD1 transcript or PCI protein in kidney cells of a subject having at least one wildtype Poly cystin 1, Transient Receptor Potential Channel Interacting (PKDP) allele, the method comprising systemically administering to a subject suffering from ADPKD caused by a mutation in a single PKD1 allele a conjugate comprising an antisense oligonucleotide (ASO) that binds to a targeted portion of the 3' untranslated region (UTR) of a wildtype PKD1 mRNA and reduces specific binding of a miR-17 family member to the 3' UTR or comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a cell penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 13, wherein the mutation results in Poly cystin 1 (PCI ) haploinsufficiency. In one example, the expression of the P D 7 transcript or PC 1 protein is increased in the kidney at least 1.1 fold

[0024] The disclosure also provides a method for increasing expression of PKD1 transcript or PCI protein in kidney cells of a subject, the method comprising systemically administering to a subject a conjugate comprising an ASO that binds to a targeted portion of the 3' untranslated region (UTR) of a Polycystic Kidney Disease 1 (PKD1) mRNA and reduces specific binding of a miR-17 family member to the 3' UTR or comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a CPP comprising the sequence set forth in SEQ ID NO: 13. In one example, the expression of the PKD1 transcript or PCI protein is increased in the kidney at least 1.1 fold. In one example, the ADPKD is caused by a mutation in a single Poly cystin 1, Transient Receptor Potential Channel Interacting (PKD1) allele in a subject resulting in Polycystin 1 (PCI) haploinsufficiency.

[0025] The disclosure additionally provides a method for increasing expression of PKD1 transcript or PCI protein in kidney cells of a subject, the method comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an ASO that binds to a targeted portion of the 3' untranslated region (UTR) of a Polycystic Kidney Disease 1 (PKDP) mRNA and reduces specific binding of a miR-17 family member to the 3' UTR or comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a CPP comprising the sequence set forth in SEQ ID NO: 13. In one example, the expression of the PKD1 transcript or PCI protein is increased in the kidney at least 1.1 fold.

[0026] In one example, the expression is increased in the medulla of the kidney.

[0027] The present disclosure also provides a method for treating ADPKD, the method comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an ASO comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a CPP comprising the sequence set forth in SEQ ID NO: 13.

[0028] In one example, a method described herein comprises administering 0.3-6.0 mg / kg of the conjugate. In one example, a method described herein comprises administering 0.3-5.0 mg / kg of the conjugate. For example, a method described herein comprises administering 1.0-2.3 mg / kg of the conjugate. For example, a method described herein comprises administering 0.3 mg / kg of the conjugate. For example, a method described herein comprises administering 1.0 mg / kg of the conjugate. For example, a method described herein comprises administering 2.0 mg / kg of the conjugate. For example, a method described herein comprises administering 2.3 mg / kg of the conjugate.

[0029] The present disclosure also provides a method for treating ADPKD, the method comprising administering to a subject suffering from ADPKD 0.3-3.5 mg / kg or 0.5-3.5 mg / kg of a conjugate comprising an ASO comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a CPP comprising the sequence set forth in SEQ ID NO: 13.

[0030] In one example, a method described herein comprises administering 0.4-5.0 mg / kg of the conjugate. For example, a method described herein comprises administering 0.4-4.0 mg / kg of the conjugate. For example, a method described herein comprises administering 0.4-2.4 mg / kg of the conjugate. For example, a method described herein comprises administering 1.2-2.4 mg / kg of the conjugate. For example, a method described herein comprises administering 0.4-1.2 mg / kg of the conjugate. For example, a method described herein comprises administering 1.2-4.0 mg / kg of the conjugate. For example, a method described herein comprises administering 2.4-4.0 mg / kg of the conjugate. For example, a method described herein comprises administering 0.4 mg / kg of the conjugate. For example, a method described herein comprises administering 1.2 mg / kg of the conjugate. For example, a method described herein comprises administering 2.4 mg / kg of the conjugate. For example, a method described herein comprises administering 4.0 mg / kg of the conjugate.

[0031] The present disclosure also provides a method for treating ADPKD. the method comprising administering to a subject suffering from ADPKD 0.4-1.2 mg / kg, 0.4-2.4 mg / kg, or 1.2-2.4 mg / kg of a conjugate comprising an ASO comprising the sequence set forth in any one of SEQ ID NOs: 1-12 and a CPP comprising the sequence set forth in SEQ ID NO: 13.

[0032] In one example, the conjugate is administered parenterally.

[0033] In one example, the conjugate is administered intravenously.

[0034] As exemplified herein following administration a therapeutically effective amount of the conjugate is detectable in a kidney and, optionally, liver of the subject.

[0035] In one example, the conjugate is detectable in the cortex or the medulla of the kidney. In one example, the conjugate is detectable to a greater degree to the cortex than the medulla of the kidney.

[0036] For example, following administration, a therapeutically effective amount of the conjugate is detectable in a cyst in a kidney of the subject.

[0037] In some examples, the therapeutically effective amount of the conjugate is detected in a kidney and, optionally in a liver in a model organism administered a dose of the conjugate that accounts for the difference in the model organism to the subject to demonstrate detection of the conjugate in the kidney and, optionally in a liver in the subject. For example, the model organism is a mouse or a non-human primate. For example, the subject is human.

[0038] In some examples, target engagement of the ASO in kidney cells of the subject is at least about 25% to 80%. In some examples, target engagement of the ASO in kidney cells of the subject is at least about 25% to 75%. In some examples, target engagement of the ASO in kidney cells of the subject is at least about 38%. In some examples, the target engagement in kidney cells of the subject is about 50%.

[0039] In some examples, the conjugate is administered in an amount sufficient to achieve target engagement of the ASO in the conjugate in kidney cells of the subject of at least about 25% to 75%. In some examples, the conjugate is administered in an amount sufficient to achieve target engagement of the ASO in the conjugate in kidney cells of the subject of at least about 38%. In some examples, the target engagement in kidney cells of the subject is about 50%.

[0040] In one example, 0.3-5.0 mg / kg of the conjugate is administered. For example, 1.0-2.3 mg / kg of the conjugate is administered. For example, a method described herein comprises administering 0.3 mg / kg of the conjugate. For example, 1.0 mg / kg of the conjugate is administered. For example, a method described herein comprises administering 2.0 mg / kg of the conjugate. For example, 2.3 mg / kg of the conjugate is administered. In one example, 0.4-5.0 mg / kg of the conjugate is administered. In one example, 0.4-4.0 mg / kg of the conjugate is administered. For example, 1.2-2.4 mg / kg of the conjugate is administered. For example, 0.4-1.2 mg / kg of the conjugate. For example, a method described herein comprises administering 0.4 mg / kg of the conjugate. For example, 1.2 mg / kg of the conjugate is administered. For example, a method described herein comprises administering 2.4 mg / kg of the conjugate.

[0041] In one example, a method described herein comprises administering multiple doses of the conjugate.

[0042] For example, the method comprises administering the conjugate to the subject at least every' four to twelve weeks.

[0043] For example, the method comprises administering the conjugate to the subject at least every' four weeks. For example, the method comprises administering the conjugate to the subject every' four weeks.

[0044] For example, the method comprises administering the conjugate to the subject at least every six weeks. For example, the method comprises administering the conjugate to the subject every' six weeks.

[0045] For example, the method comprises administering the conjugate to the subject at least every eight weeks. For example, the method comprises administering the conjugate to the subject every eight weeks.

[0046] For example, the method comprises administering the conjugate to the subject at least every 12 weeks. For example, the method comprises administering the conjugate to the subject every 12 weeks.

[0047] In one example, the CPP in the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 13. In some examples, each amino acid in the CPP is a D amino acid (other than glycine for which there is no D amino acid).

[0048] In one example, the nucleotide sequence of the ASO in the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 1.

[0049] In some examples, the antisense oligonucleotide comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage or wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino linkage. For example, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0050] In one example, the conjugate additionally comprises a linker between the CPP and the antisense oligonucleotide. For example, the linker is a peptide linker, e.g., a flexible peptide linker. Suitable peptide linkers are described herein.

[0051] In one example, the conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1.

[0052] In one example, the conjugate consists of: (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13; and

[0053] (ii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide.

[0054] In one example, the C-terminus of the CPP is linked to the secondary amine at the 3' end of the antisense oligonucleotide. For example, the carboxylic acid C-terminus of the CPP is linked to the secondary amine at the 3' end of the antisense oligonucleotide via a linker.

[0055] In one example, the conjugate consists of:

[0056] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids;

[0057] (ii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide.

[0058] In one example, the conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0059] In one example, the conjugate consists of:

[0060] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13;

[0061] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16; and

[0062] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0063] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the antisense oligonucleotide.

[0064] In one example, the conjugate consists of: (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid):

[0065] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0066] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide.

[0067] In one example, the conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to a PMO comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 1. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0068] In one example, the conjugate consists of:

[0069] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13;

[0070] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16; and

[0071] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0072] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the PMO.

[0073] In one example, the conjugate consists of:

[0074] (i) a CPP the amino acid sequence of w hich consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid):

[0075] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide.

[0076] The present disclosure also provides a method for treating autosomal dominant polycystic kidney disease (ADPKD), the method comprising intravenously administering to a subject suffering from ADPKD 0.4-4.0 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0077] The present disclosure also provides a method for treating autosomal dominant polycystic kidney disease (ADPKD), the method comprising intravenously administering to a subject suffering from ADPKD 0.4-2.4 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0078] In another example the method comprises intravenously administering to a subject suffering from ADPKD 0.4-1.2 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0079] In another example the method comprises intravenously administering to a subject suffering from ADPKD 1.2-2.4 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0080] In another example the method comprises intravenously administering to a subject suffering from ADPKD 1.2-4.0 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0081] In another example the method comprises intravenously administering to a subject suffering from ADPKD 2.4-4.0 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0082] In one example the method comprises intravenously administering to a subject suffering from ADPKD 0.4 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in any one of SEQ ID NOs: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0083] In another example the method comprises intravenously administering to a subject suffering from ADPKD 1.2 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in SEQ ID NO: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13. In another example the method comprises intravenously administering to a subject suffering from ADPKD 2.4 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in SEQ ID NO: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0084] In another example the method comprises intravenously administering to a subject suffering from ADPKD 4.0 mg / kg of a conjugate comprising an ASO consisting of the sequence set forth in SEQ ID NO: 1 and a CPP consisting of the sequence set forth in SEQ ID NO: 13.

[0085] The present disclosure additionally provides a conjugate comprising or consisting of an ASO the nucleotide sequence of which consists of the sequence set forth in of SEQ ID NO: 1 and a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13.

[0086] In one example, the conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0087] In one example, the conjugate consists of:

[0088] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13;

[0089] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16; and

[0090] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0091] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the antisense oligonucleotide.

[0092] In one example, the conjugate consists of:

[0093] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid): (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0094] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide.

[0095] In one example, the conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to a PMO comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid).

[0096] In one example, the conjugate consists of:

[0097] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13;

[0098] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16; and

[0099] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide. In one example, the CPP and / or the linker includes only D-amino acids (other than glycine for which there is no D amino acid). In one example, the CPP and the linker includes only D-amino acids (other than gly cine for which there is no D amino acid).

[0100] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary’ amine at the 3' end of the PMO.

[0101] In one example, the conjugate consists of:

[0102] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0103] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0104] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide. The disclosure additionally provides a pharmaceutical composition comprising a conjugate comprising or consisting of an ASO the nucleotide sequence of which consists of the sequence set forth in any one of SEQ ID NOs: 1 and a cell penetrating peptide (CPP) the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13.

[0105] The disclosure also provides a pharmaceutical composition comprising an ASO which binds to a miR-17 binding site in a 3' UTR of aPKDl gene transcript in a cell, wherein the pH of the composition is between 5.0 and 7.0.

[0106] The disclosure also provides a phamiaceutical composition comprising an ASO which binds to a miR-17 binding site in a 3' UTR of aPKDl gene transcript in a cell, wherein the pH of the composition is between 5.5 and 6.5.

[0107] In one example, the ASO comprises a backbone modification. For example, the backbone modification is a phosphorothioate linkage or a phosphorodiamidate linkage or a phosphorodiamidate morpholino linkage. For example, the ASO is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0108] The disclosure also provides a pharmaceutical composition comprising a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of aPKDl gene transcript in a cell and a CPP, wherein the pH of the composition is between 5.5 and 6.5.

[0109] The disclosure also provides a pharmaceutical composition comprising a conjugate described herein wherein the pH of the composition is between 5.5 and 6.5.

[0110] In one example, the pH of the pharmaceutical composition is between 5.5 and 6.5. For example, the pH of the pharmaceutical composition is 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the pH of the pharmaceutical composition is 6.0.

[0111] The disclosure also provides a pharmaceutical composition comprising an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a buffer.

[0112] In one example, the ASO comprises a backbone modification. For example, the backbone modification is a phosphorothioate linkage or a phosphorodiamidate linkage or a phosphorodiamidate morpholino linkage. For example, the ASO is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0113] The disclosure also provides a pharmaceutical composition comprising:

[0114] (i) a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a CPP; and

[0115] (ii) a buffer.

[0116] The disclosure also provides a pharmaceutical composition comprising a conjugate described herein and a buffer.

[0117] In one example, the buffer is a physiological buffer.

[0118] In one example, the buffer is effective to maintain the pH of the composition at a pH of between 5.5 and 6.5. For example, the buffer is effective to maintain the pH of the composition at a pH of 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the buffer is effective to maintain the pH of the composition at a pH of 6.0.

[0119] Reference herein to a pH will be understood to mean the target pH or the pH in the middle of an acceptable range of pHs, e.g., as indicated in a release criterion for a drug. For example, a pH of 6.0 encompasses a pH of 6.0±0.2 or 0.3.

[0120] In one example, the pH of the pharmaceutical composition is between 5.5 and 6.5. For example, the pH of the pharmaceutical composition is 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the pH of the pharmaceutical composition is 6.0.

[0121] In one example, the buffer is a phosphate buffer.

[0122] In one example, the pharmaceutical composition additionally comprises a tonicity modifier.

[0123] In one example, the tonicity modifier is sodium chloride.

[0124] The disclosure provides a pharmaceutical composition comprising:

[0125] (i) a conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16, wherein the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0126] (ii) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0127] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0128] The disclosure additionally provides a pharmaceutical composition comprising:

[0129] (A) a conjugate consisting of:

[0130] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0131] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0132] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide; and

[0133] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0134] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the antisense oligonucleotide. In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0135] The disclosure additionally provides a pharmaceutical composition comprising:

[0136] (A) a conjugate consisting of:

[0137] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0138] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0139] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide;

[0140] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0141] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0142] The disclosure provides a pharmaceutical composition comprising:

[0143] (i) a conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13. linked to a PMO comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the PMO by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16, wherein the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0144] (ii) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0. In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0145] The disclosure additionally provides a pharmaceutical composition comprising:

[0146] (A) a conjugate consisting of:

[0147] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0148] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0149] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the PMO; and (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0. In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C -terminus of the linker is linked to the secondary amine at the 3' end of the PMO.

[0150] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0151] The disclosure additionally provides a pharmaceutical composition comprising:

[0152] (A) a conjugate consisting of:

[0153] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0154] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0155] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the PMO;

[0156] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0157] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0158] In one example, the pharmaceutical composition is lyophilized.

[0159] In one example, the lyophilized composition has:

[0160] (i) the conjugate at a purity of at least 90%, as determined by high performance liquid chromatography (HPLC);

[0161] (ii) a total impurity content of less than 10%, as determined by HPLC; and / or

[0162] (iii) a water content of less than 5%, as determined by Coulometric Karl Fischer (CKF) assay.

[0163] In one example, the purity of the conjugate is at least 91%. In one example, the purity of the conjugate is at least 91.5%.

[0164] In one example, the impurity content is less than 9%. In one example, the impurity content is less than 8.6%.

[0165] In one example, the total impurity content, purity and / or water content of the conjugate is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 12 months, or at least 18 months at a temperature of: -20°C ± 5°C and / or 5°C ± 3°C.

[0166] In one example, the composition is a liquid formulation.

[0167] In one example, the composition is or was previously reconstituted from a lyophilized formulation having:

[0168] (i) the conjugate at a purity of at least 90%, as determined by high performance liquid chromatography (HPLC); (ii) a total impurity content of less than 10%, as determined by HPLC; and / or

[0169] (iii) a water content of less than 5%, as determined by Coulometric Karl Fischer (CKF) assay.

[0170] In one example, the purity of the conjugate is at least 91%. In one example, the purity of the conjugate is at least 91.5%.

[0171] In one example, the impurity content is less than 9%. In one example, the impurity content is less than 8.6%.

[0172] In one example, the total impurity content, purity and / or water content of the conjugate is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 12 months, or at least 18 months at a temperature of: -20°C ± 5°C and / or 5°C ± 3°C.

[0173] In one example, the composition is a liquid or reconstituted composition and has: (i) a total impurity content of no more than 7%, as determined by Ultra Performance Liquid Chromatography (UPLC);

[0174] (ii)the conjugate at a purity of at least 92%, as determined by UPLC; and / or

[0175] (iii) an osmolality in the range of 300 mOsm / kg to 310 mOsm / kg.

[0176] In one example, the total impurity content is no more than 6.5%. In one example, the total impurity content is no more than 6.1%.

[0177] In one example, the purity of the conjugate is at least 93.0%. In one example, the purity of the conjugate is at least 93.9%.

[0178] In one example, the osmolality is 303 mOsm / kg or 306 mOsm / kg. Reference herein to an osmolality will be understood to mean the target osmolality or the osmolality in the middle of an acceptable range of osmolalities, e.g., as indicated in a release criterion for a drug. For example, an osmolality is 303 mOsm / kg encompasses an osmolality of 303±3 or 2.

[0179] In one example, the total impurities, purity' and / or osmolality is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, or at least 12 months at a temperature of 5°C ± 3°C.

[0180] In one example, the pharmaceutical composition comprises the conjugate at a purity of at least 90%, as determined by HPLC following incubation at 50°C for three days.

[0181] In one example, the pharmaceutical composition comprises the conjugate at a purity of at least 88%, as determined by HPLC following incubation at 50°C for six days.

[0182] In one example, the pharmaceutical composition is a liquid composition or reconstituted composition comprising the conjugate at a concentration of 10 mg / mL to 30 mg / mL. For example the pharmaceutical composition is a liquid composition or reconstituted composition comprising the conjugate at a concentration of 15 mg / mL to 25 mg / mL.

[0183] For example, the pharmaceutical composition is a liquid composition or reconstituted composition comprising the conjugate at a concentration of 30 mg / mL. For example, the pharmaceutical composition is a liquid composition or reconstituted composition comprising the conjugate at a concentration of 20 mg / mL.

[0184] The disclosure also provides a container comprising a conjugate or pharmaceutical composition disclosed herein.

[0185] In one example, the container is a vial and / or a syringe.

[0186] For example, the composition is supplied in a glass vial.

[0187] In one example, the vial comprises the composition having a volume in the range of 5 mL to 15 mL. For example, the composition has a volume in the range of 7 mL to 12 mL. For example, the formulation has a volume of 10 mL.

[0188] In one example, the composition comprises the conjugate at a concentration of 10 mg / mL. In one example, the composition comprises the conjugate at a concentration of 10 to 30 mg / mL. In one example, the composition comprises the conjugate at a concentration of 15 to 25 mg / mL. In one example, the composition comprises the conjugate at a concentration of 20 mg / mL.

[0189] The present disclosure additionally provides a container comprising an amount of a conjugate disclosed herein, wherein the container comprises sufficient conjugate for administration at a dose of 10 mg / mL to 30 mg / mL. For example, the disclosure additionally provides a container comprising an amount of a conjugate disclosed herein, wherein the container comprises sufficient conjugate for administration at a dose of 15 mg / mL to 25 mg / mL. For example, the disclosure additionally provides a container comprising an amount of a conjugate disclosed herein, wherein the container comprises sufficient conjugate for administration at a dose of 20 mg / mL.

[0190] In one example, the conjugate is lyophilized.

[0191] In one example, the container comprises a liquid or reconstituted formulation comprising the conjugate.

[0192] Brief description of the Drawings

[0193] Figure 1 is a series of photomicrographs showing peptide PMO (PPMO) localisation in the in kidneys of non-human primates from a dose range finding study

[0194] Non-human primates received either vehicle control or PPMO through intravenous infusion. After 28 days, kidneys were collected, fixed in formalin, and prepared for analysis using miRNAScope assays. This involved hybridising with a PPMO-specific probe, followed by signal amplification and visualization. Images captured at 20x magnification displayed PPMO staining in dark grey and kidney morphology in light grey. The results included representative transverse kidney sections for both treatment groups, with additional zoomed-in images of specific regions in the cortex and medulla. Figure 2 incudes a series of graphical representations showing semi-quantitative analysis of PPMO signal in the kidney of non-human primates from a dose range finding study Images of all animals mentioned in Example 1 were analysed using a semi-quantitative automated histological scoring method to assess the optical density (OD) of probe staining in both the kidney cortex and medulla (Figure 2A). The OD measurements from treated animals were normalised against those from vehicle-treated animals. The ratio of OD between the cortex and medulla in treated animals is shown in Figure 2B.

[0195] Figure 3 includes a table and a graphical representation showing tissue concentrations of PPMO in non-human primates from a dose range finding study

[0196] Quantification of a PPMO with a sequence corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO:1) in kidneys of non-human primates. Non-human primates were administered either vehicle control or PPMO at 3, 10 or 30 mg / kg via intravenous infusion, and various organs including kidney, liver, pancreas, heart, and skeletal muscle were collected. PPMO levels in tissue lysates were quantified using a qualified ELISA method, normalised to the amount of tissue processed to calculate the PPMO tissue concentration. Drug concentrations in the kidney were evaluated at 48 hours (3 mg / kg group only) and 28 days posttreatment (Figure 3A). Additionally, drug concentrations in the kidney, liver, pancreas, heart, and skeletal muscle were analysed after PPMO administration at 3 mg / kg PPMO (Figure 3B).

[0197] Figure 4 includes a table and a graphical representation showing PPMO concentration in tissues in non-human primates from a pharmacokinetics study

[0198] Quantification of a PPMO with a sequence corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO: 1) in kidneys of non-human primates. Animals were intravenously infused with PPMO at doses of 10 or 30 mg / kg. Tissue samples from various organs including the kidney cortex, kidney medulla, liver, pancreas, and skeletal muscle were collected at specific doses and time points. The concentrations of PPMO in the tissue lysates were measured using a qualified ELISA method and normalized based on the amount of tissue processed, allowing for the calculation of PPMO tissue concentration. Drug concentrations in kidney cortex and medulla samples were assessed 24 hours post-treatment for all groups and at 56 days post-treatment for the 10 mg / kg group (Figure 4A). Additionally, drug concentrations in the kidney cortex, kidney medulla, liver, pancreas, and skeletal muscle were measured at designated time points after dosing with 10 mg / kg PPMO (Figure 4B)

[0199] Figure 5 is a graphical representation showing effect of PPMO on PCI protein levels in kidneys of non-human primates from a non-GLP pharmacokinetics study

[0200] Selected samples from animals in Example 4 were analysed to evaluate the PPMO effect on protein levels. Kidney medulla protein levels from vehicle or PPMO-treated animals were assessed using a western blot assay. Proteins were extracted from day-28 post-treatment samples using RIPA buffer. Total protein was quantified with a BCA protein kit, run on gels, and proteins were transferred to a nitrocellulose membrane via wet transfer before staining for total protein and PCI protein. Blots were imaged on an Odyssey Imager, and quantitative analysis was performed using Image Studio Ver 5.5. The raw fluorescence signal for PCI protein was normalised to the total protein and expressed as fold-change relative to vehicle-treated samples. Data from n=2 western blot assays are presented as mean ± standard error of the mean.

[0201] Figure 6 includes a series of photomicrographs showing PPMO localisation the in kidneys of normal and Pkdl deficient mice

[0202] PPMO distribution in healthy and cystic kidneys was assessed using a tamoxifen-inducible conditional knock-out mouse model otPkdl. Mice without tamoxifen induction are healthy, while treatment with tamoxifen leads to Pkdl gene deletion and ADPKD development. At PND70, both healthy and diseased mice were treated with a PPMO with a sequence corresponding to PKD1 H46 3’UTR(+604+628) (SEQ ID NO:1) and kidneys were collected for analysis three days later. Kidney were fixed in 10% formalin for 24 hours and processed for paraffin embedding. miRNAScope assays, using an ACD Bio kit, involved hybridisation with a PPMO-specific probe, followed by signal amplification and visualization. Images were captured with a 3DHISTECH Pannoramic Flash 250 III Slide Scanner at 20x magnification, showing PPMO staining in dark grey and hematoxylin counter-staining in light grey. Zoomedin images of three regions from the cortex and medulla are also included.

[0203] Figure 7 Target engagement and PCI protein upregulation in HEK293 cells following PPMO treatment

[0204] A PMO with sequences corresponding to PKD1 H46 3'UTR(+604+628) (SEQ ID NO: 1 ) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). The data shows that engagement of the PKD1 mRNA 3' UTR by the PPMO leads to increased PCI protein levels in HEK293 cells.

[0205] (A) Digital droplet PCR (ddPCR) quantification of PKD1 mRNA expression in HEK293 cells treated with 60 pM PPMO (SEQ ID NO: 1 conjugated to SEQ ID NO: 13) for 3 days. Target engagement was assessed by inhibition of reverse transcription due to PMO binding to the 3' UTR of PKD1 mRNA. PKD1 expression was normalized to DHX57 and reported as fold-change relative to untreated cells. A non-targeting control oligonucleotide (GTC CTR) was used as a negative control. Treatment resulted in 54% target engagement.

[0206] (B) Western blot analysis of PCI protein levels in lysates collected at day 3. PCI was probed using an anti-PCl antibody and detected by infrared fluorescence. Signal was normalized to total protein and reported as fold-change relative to untreated control. A 1.75-fold increase in PCI expression was observed. RGLS4326 was included as a positive control.

[0207] Figure 8 PPMO target engagement and PCI induction in ADPKD patient-derived proximal tubule cyst cells (PKD1 p. Q2556*)

[0208] A PMO with sequences corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO:1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). Taken together, the data shown indicates that PPMO-mediated engagement of the PKD1 3' UTR is sufficient to enhance PCI protein expression in ADPKD patient-derived proximal tubule cyst cells (PKD1 p. Q2556*).

[0209] (A) ddPCR analysis of PKD1 mRNA in patient-derived cystic cells treated with 60 uM PPMO for 2 days. Target engagement was measured by inhibition of RT, normalized to DHX57, and reported relative to untreated controls. A 53% target engagement was observed. GTC CTR served as a negative control.

[0210] (B) Western blot analysis of PCI protein from the same samples collected at day 5. A 1.2-fold increase in PCI protein expression was detected relative to untreated controls. Quantification was normalized to total protein. RGLS4326 was included as a positive control.

[0211] Figure 9 Effect of PPMO treatment on PKD1 target engagement and PCI protein levels in ADPKD iPSCs (PKD1 p. A1458fs)

[0212] A PMO with sequences corresponding to PKD1 H463UTR(+604+628) (SEQ ID NO: 1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). The data demonstrates that PPMO targeting of the PKD1 3' UTR effectively increases PCI protein levels in ADPKD iPSCs (PKD1 p. A1458fs) cells.

[0213] (A) ddPCR quantification of PKD1 mRNA expression in iPSC-derived cells treated with 30 pM PPMO for 2 days. A 35% target engagement was observed, measured by reduced RT efficiency due to PPMO binding to the PKD1 3' UTR. Values are normalized to DHX57 and reported as fold-change versus untreated cells. GTC CTR was used as a negative control.

[0214] (B) PCI protein levels measured by Western blot in samples collected at day 5. Treatment resulted in a 1.5-fold increase in PCI protein, normalized to total protein. RGLS4326 was included as a positive control for PCI upregulation.

[0215] Figure 10 PPMO-mediated target engagement in a 3D cyst model derived from ADPKD patient kidneys

[0216] Cystic epithelial cells isolated from kidneys of ADPKD patients (donor lines huPKD04 and huPKDll) were cultured in a 3D matrix, where they spontaneously formed cysts. Cells were treated immediately after seeding with 20 pM PPMOs, composed of a PMO corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO:1) conjugated to a cell-penetrating peptide (SEQ ID NO: 13). After 6 days, total RNA was extracted and PKD1 transcript levels were quantified by TaqMan droplet digital PCR (ddPCR) using probe Hs00947394_gl. Expression was normalized to the housekeeping gene DHX57 (probe Hs00376574_ml) and reported as foldchange relative to untreated controls. A non-targeting oligonucleotide (GTC CTR) served as a negative control. PPMO treatment led to a reduction in PKD1 expression ranging from 38% to 65%, demonstrating target engagement across both donor-derived cyst models.

[0217] Figure 11 Structure of conjugate formula I

[0218] A graphical illustration of structural formula I a conjugate in which a CPP (SEQ ID NO: 13) is linked to a PMO (SEQ ID NO: 1) through a flexible peptide linker (SEQ ID NO: 16).

[0219] Figure 12 Mean Plasma conjugate (formula I) concentration-time profiles - 0.4, 1.2 and 2.4 mg / kg human dose cohorts

[0220] Line graphs of concentration-time profiles for subjects dosed in single ascending dose (SAD) study at 0.4 mg / kg, 1.2 mg / kg, and 2.4 mg / kg doses.

[0221] Figure 13 Structure of conjugate formula “PPMO 10”

[0222] A graphical illustration of the structural formula of another conjugate of the disclosure, in which a CPP and oligonucleotide are linked through a D-azidolysine linkage obtained through strain promoted azide-alkyne cycloaddition (SPAAC) conjugation.

[0223] Detailed Description

[0224] General

[0225] 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 groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes 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.

[0226] Each example of the present disclosure described herein is to be applied mutatis mutandis to each and every other example unless specifically stated otherwise.

[0227] Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0228] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0229] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology'. Such techniques are described and explained throughout the literature in sources such as Perbal 1984, Sambrook et al., 2001, Brown (editor) 1991, Glover and Hames (editors) 1995 and 1996, Ausubel et al. including all updates until present, Coligan et al. (editors) (including all updates until present), Maniatis et al. 1982, Gait (editor) 1984, Hames and Higgins (editors) 1984, Freshney (editor) 1986.

[0230] 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 meanings or for either meaning.

[0231] The term '‘about”, unless stated to the contrary, refers to + / - 20%, more preferably + / -10%, of the designated value. For the avoidance of doubt, the term "about” followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, “about 10” also encompasses 10 exactly).

[0232] 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.

[0233] The term “antisense oligonucleotide” “antisense oligomer” or “ASO,” as used herein, encompasses oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary sequence on a target RNA transcript, including, but not limited to, those that do not comprise a sugar moiety, such as in the case of a peptide nucleic acid (PNA). For example, the ASO is an ASO that is resistant to nuclease cleavage or degradation.

[0234] The phrase “binds to a targeted portion” or “binds within a targeted portion,” in reference to an ASO, as used herein, refers to specific hybridization between the ASO nucleotide sequence and a target nucleotide sequence that is complementary within the ranges set forth herein. In some examples, specific hybridization occurs where, under ex vivo conditions, the hybridization occurs under high stringency conditions. By "high stringency conditions" is meant that the ASO, under such ex vivo conditions, hybridize to a target sequence in an amount that is detectably stronger than non-specific hybridization. High stringency conditions, then, are conditions that distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 1-5 bases) that matched the probe. Such small regions of complementarity are more easily melted than a full-length complement of 12-17 or more bases, and moderate stringency hybridization makes them easily distinguishable. 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 the equivalent, at temperatures of about 50-70 °C. The skilled person will appreciate that under in vivo conditions, the specificity of hybridization between an ASO and its target sequence is defined in terms of the level of complementarity between the ASO and the target sequence to which it hybridizes within a cell.

[0235] The term “target engagement” in reference to an “ASO” or “conjugate,” as used herein, refers a proportion of PKD1 3’ UTR target sequence site copies hybridized with the ASO or constituent ASO in the conjugate. In some examples, such target engagement is determined based on a level of quantitative reverse transcription PCR (qRT-PCR) observed.. Thus, in an RNA sample obtained from a subject or cells previously exposed to the ASO, the level of RT-PCR amplification will be reduced, as the presence of the ASO on the target 3’ UTR sequence of the mRNA interferes with reverse transcription by physically blocking the reverse transcriptase from accessing or extending through the target region.. Thus, the lower the level of RT-PCR amplification observed relative to the level of RT-PCR in an RNA sample never exposed to the ASO. the higher the level of target engagement.

[0236] The term “peptide” is intended to include compounds composed of amino acid residues linked by amide bonds. A peptide may be natural or unnatural, ribosome encoded or synthetically derived. Typically, a peptide will consist of between 2 and 200 amino acids. For example, the peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within said range(s). The peptide may 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.

[0237] Peptides, as referred to herein, include "inverso" peptides in which all L-amino acids are substituted with the corresponding D-amino acids, "retro-inverso" peptides in which the sequence of amino acids is reversed and all L-amino acids are replaced with D-amino acids.

[0238] Peptides may comprise amino acids in both L- and / or D-form. For example, both L-and D-forms may 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, such as natural amino acids. In some examples the amino acids within the peptide sequence are a combination of L-and D-form. Further, peptides may comprise unusual, but naturally occurring, amino acids including, but not limited to, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle), 3 -nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr). Peptides may also incorporate unnatural amino acids including, but not limited to, homo amino acids, N-methyl amino acids, alpha-methyl amino acids, beta (homo) amino acids, gamma amino acids, and N-substituted glycine. Peptides may be linear peptides or cyclic peptides.

[0239] The term ’protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical bond or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0240] Percentage amino acid sequence identity with respect to a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues 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 may be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix ’Blosum62" is utilized for amino acid sequences and the default matrix.

[0241] The term ‘‘cell penetrating peptide'’ (CPP) refers to a peptide that is capable of crossing a cellular membrane. In one example, a CPP is capable of translocating across a mammalian cell membrane and entering into a cell. In another example, a CPP may direct a conjugate to a desired subcellular compartment. Thus, a CPP may direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A CPP may be translocated across the membrane with its amino acid sequence complete and intact, or alternatively partially degraded.

[0242] A CPP may direct a molecule of interest or compound, such as an ASO disclosed herein, from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a CPP may direct a molecule of interest across the, epithelial, endothelial, basement membrane, trans-mucosal, cardiovascular, skin, gastrointestinal and / or pulmonary barriers. In some examples the CPP is preferentially targeted to or taken up by the kidneys. Compositions for Increasing AD 7 mRNA and Polvcvstin-1 Protein Levels MicroRNAs (miRNAs) are a family of short (19 to 23 nucleotide) non-coding single stranded labile RNAs. Although they can bind to any part of target mRNA. their main mode of action is to bind to complimentary RNA sequences in the 3' untranslated region (3' UTR) and regulate gene expression by stimulating either mRNA degradation or translational repression. Both mechanisms lead to diminished expression of the target gene. In the case of the Polycystic Kidney Disease 1 (PKDP) gene that encodes Polycystin 1 protein, miRNAs that bind to the 3' UTR and of the encoding mRNA transcript include miR-17 family miRNAs (e.g, miR-17-5p, miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-93-5p).

[0243] Conjugates disclosed herein comprise an ASO that binds to a targeted portion of the 3' UTR of a wildtype PKD1 allele mRNA in a ADPKD1 subject that is haploinsufficient for PCI and heterozygous for a wildtype PKD1 allele encoding functional PCI and a mutated PKD1 allele that produces non-functional PCI or no PCI, wherein binding of the antisense oligonucleotide to the targeted portion increases the level of PKD1 mRNA and / or Polycystin 1 protein. In some examples the ASO hybridizes to a targeted portion of the 3' UTR of wildtype PKD1 mRNA, whereby one or more miRNAs are unable to hybridize to their specific target sequences and consequently, increased levels of PKD1 mRNA and functional Poly cystin 1 protein.

[0244] For reference, the sequence of the canonical human PKD1 mRNA transcript (“PKD-201”) is publicly available through the online Ensembl database under record ENST00000262304.9.

[0245] Antisense Oligonucleotides (ASOs)

[0246] In some examples of the conjugates and methods described herein, ASOs have a sequence that is completely complementary across its length to the target sequence or a sequence near complementarity (e.g, sufficient complementarity to bind the target sequence and interfere with miRNA binding at a wildtype PKD1 mRNA 3' UTR binding site). ASOs are designed so that they bind (hybridize) to a target RNA sequence (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Selection of suitable sequences for ASOs generally avoids, where possible, similar nucleic acid sequences in other (i.e., off-target) locations in the genome or in cellular mRNAs or miRNAs, such that the likelihood the ASO will hybridize at such sites is limited.

[0247] In some examples, ASOs ’‘specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of the PKD1 mRNA 3' UTR. At a given ionic strength and pH, the Tmis the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.

[0248] ASO sequences are “complementary” to their target sequences when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides of complementary sequence. Complementarity is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The nucleotide sequence of an ASO need not be 100% complementary to that of its target nucleic acid to hybridize. In certain examples, the nucleotide sequences of ASOs in the compositions disclosed herein can be 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% sequence complementary to the nucleotide sequence of the targeted portion of an RNA transcript over the length of the ASO nucleotide sequence. For example, an ASO in which 18 of 20 nucleotides of ASO or AR sequence are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In such an example, the remaining non-complementary nucleotides of the ASO could be clustered together or interspersed with complementary nucleotides and need not be contiguous. Complementarity of an ASO sequence to a target nucleotide sequence (expressed as ‘'percent complementarity ” to its target sequence; or ‘'percent identity” to its reverse complement sequence) can be determined routinely using algorithms known in the art, as exemplified in the BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul, et al., 1990, J. Mol. Biol., 215:403-410; Zhang et al., 1997, Genome Res., 7:649-656).

[0249] In some examples, an ASO does not hybridize to all nucleotides in a target sequence and the nucleotide positions at which it does hybridize may be contiguous or non-contiguous. ASOs may hybridize over one or more segments of a 3' UTR region of a mRNA, such that intervening or adjacent segments are not involved in the hybridization event (e.g, a loop structure or hairpin structure may be formed).

[0250] The ASOs for use in the conjugates described herein may be of any length suitable for specific hybridization to a target sequence. In some examples, the nucleotide sequence of the ASOs consist of 8 to 50 nucleotides. For example, the ASO 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 ASOs consist of more than 50 nucleotides, but no more than 100 nucleotides in length. In some examples, the ASO or AR nucleotide sequence is from 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 ASOs or ARs are 20 nucleotides in length. In some examples, the nucleotide sequence of the ASO or AR nucleotide is 25 nucleotides in length.

[0251] In some examples for each occurrence of “G” in an ASO disclosed herein, the “G” is guanosine or inosine. In some examples for each occurrence of “T” in an ASO sequence disclosed herein, the “T” is any one of: thymidine, inosine, uracil, or an isomeric or modified form of uracil (e.g., pseudouridine or Nl-methyl-pseudouridine). In some examples for each occurrence of “C” in an ASO sequence disclosed herein, the C is cytosine or a modified form of cytosine (e.g., 5 -methyl cytosine).

[0252] In some examples a suitable the nucleotide sequence of the ASO is described in, e.g., PCT / AU2024 / 050616 and comprises the sequence of any one of SEQ ID NOs:l-12. In some examples the nucleotide sequence of the ASO consists of the sequence of any one of SEQ ID NOs: 1-12. In some examples the nucleotide sequence of the ASO or AR comprises or consists of the sequence of SEQ ID NO: 1.

[0253] ASO Chemistry and Modifications

[0254] The ASOs used in the compositions described herein may comprise naturally-occurring nucleotides, nucleotide analogues, modified nucleotides, or any combination thereof. The term “naturally occurring nucleotides’' includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides with modified or substituted sugar groups and / or having a modified backbone. In some examples, all the nucleotides of an ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the compositions and methods described herein are known in the art as disclosed in, e.g., in 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 Rev. Drug Disc., 19:673-694.

[0255] One or more nucleotides of an ASO may be any naturally-occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine, uracil and inosine, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a 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.

[0256] ASOs include a “backbone” structure, that refers to the connection between nucleotides / monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of adjacent nucleotides. Suitable types of backbone linkages for the ASOs described herein include, but are not limited to, phosphodiester. phosphorothioate, phosphorodithioate, phosphorodiamidate, phosphoroselenoate, phosphorodiselenoate. phosphoroanilothioate, phosphoraniladate, 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), Biomedicines, 9:503.

[0257] In some examples, the backbone structure of the ASO does not contain phosphorous-based linkages, but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.

[0258] In some examples, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In other examples, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is controlled and is not random. For example, U. S. Pat. No. 9,605,019 describes methods for independently selecting the handedness of chirality at each phosphorous atom in an oligonucleotide. In some examples, an ASO used in the conjugates and methods provided herein, including, but not limited to, the ASOs the sequences of which are disclosed herein as SEQ ID NOs:l-12 is an ASO having phosphodiester intemucleotide linkages that are not random. In some examples, a conjugate or conjugate used in the methods disclosed herein comprises a pure diastereomeric ASO. In other examples, the conjugate comprises an ASO that has 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%.

[0259] In some examples, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. In some examples, an 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 Sp, or about 100% Rp.

[0260] In some examples, the ASOs described herein contain a sugar moiety that comprises 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' substitutions such as 2'-O-modifications, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium. 2'-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some examples, the sugar moiety modification is selected from among 2'-O-Me, 2'F, and 2'MOE. In other examples, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some examples the sugar analogue contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some examples, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some examples, the sugar moiety comprises 2'4' -constrained 2'-O-methyloxyethyl (cMOE) modifications. In some examples, the sugar moiety comprises cEt 2', 4' constrained -O ethyl BNA modifications. In other examples, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some examples, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some examples, the sugar moiety comprises 2'-O-(2-N-methylcarbamoylethyl) (MCE). Modifications are known in the art as exemplified in Jarver, et al., 2014, Nucleic Acid Therapeutics, 24(1): 37-47.

[0261] In some examples, each constituent nucleotide of the ASO is modified in the same way, e.g., every linkage of the backbone of the ASO comprises a phosphorothioate linkage, or each ribose sugar moiety comprises a 2'-O-methyl modification. In other examples, a combination of different modifications is used, e.g., an ASO comprising a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).

[0262] In some examples, the ASO comprises one or more backbone modifications. In some examples, the ASO comprises one or more sugar moiety modification. 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).

[0263] In some preferred examples, the ASO comprises or is a phosphorodiamidate morpholino (PMO).

[0264] The skilled person in the art will appreciate that ASOs may be modified, in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. In some examples, an 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 sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of an ASO into a cell and / or particular subcellular compartments; alter the pharmacokinetics or pharmacodynamics of the ASO; and / or modulate the half-life of the ASO in vivo.

[0265] In some examples, the ASOs comprise one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides that have been shown to confer significantly enhanced resistance of ASOs to nuclease degradation and increased bioavailability. Methods for synthesis and chemical modification of ASOs, as well as synthesis of ASO conjugates is well known in the art, and such ASOs are available commercially.

[0266] In some examples, the conjugates disclosed herein include ASOs that are linked to a functional moiety. In some preferred examples the functional moiety is a delivery moiety.

[0267] Suitable delivery moieties include CPPs.

[0268] In some examples, the delivery moiety includes a cell-penetrating peptide (CPP).

[0269] Cell Penetrating Peptides (CPP)

[0270] The conjugate of the present disclosure comprises a CPP comprising the sequence set forth in SEQ ID NO: 13. The CPP can consist of the sequence set forth in SEQ ID NO: 13.

[0271] In some examples the amino acid sequence of a CPP provided herein includes at least one D-amino acid. In some examples the amino acid sequence of a CPP includes only D-amino acids (other than glycine, for which there is no D form). For example, the amino acid sequence of the CPP is D-argnyl-D-arginyl-D-Seryl-D-arginyl-D-threonyl-D-alanyl-D-arginyl-D-alanyl-glycine-D-arginyl-D-prolyl-glycine-D-arginyl-D-asparaginyl-D-seryl-D-seryl-D-arginyl-D-prolyl-D-seryl-D-alanyl-D-prolyl-D-arginyl.

[0272] In other examples, a CPP includes at least one L-amino acid. In some examples, the CPP includes only L-amino acids.

[0273] In some examples the amino acid sequence of a CPP provided herein is the inverse sequence of any of the CPP amino acid sequence enumerated herein.

[0274] In some examples the amino acid sequence of a CPP provided herein is the retro-inverso sequence of any of the CPP amino acid sequence enumerated herein.

[0275] A CPP may direct a molecule of interest, such as an antisense oligonucleotide disclosed herein, from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a CPP may direct a molecule of interest across the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal and / or pulmonary barriers.

[0276] CPP Conjugates

[0277] The term “conjugate,” as used herein, refers to a CPP that is linked (covalently or non-covalently) to a ASO as described herein.

[0278] In some examples, the CPP is covalently conjugated to the ASO.

[0279] In some examples, the CPP is directly conjugated to the ASO.

[0280] In some examples, a CPP is linked to an ASO through a linker, e.g., a flexible linker linking the CPP and a heterologous amino acid sequence such a peptide or protein. Examples of flexible linkers include, but are not limited to, GGGGS, GGGGSGGGGS (SEQ ID NO: 14), GAS, GGG, GSG, GTG, GGTAGSTGG (SEQ ID NO: 15), GASGGASG (SEQ ID NO: 16) and GASG. Other examples of such flexible linkers are known in the art as described in, e.g., Chen et al (2013), Adv Drug Deliv Rev., 65(10): 1357-1369. In one example, the amino acid sequence of the linker only includes D-amino acids. In another example, a linker is conjugated to the ASO through Strain promoted azide-alkyne cycloaddition (SPAAC) conjugation, where the linking moiety can be a D-azidolysine, as exemplified in PMO10 (illustrated in Fig. 13).

[0281] In one example, the carboxylic acid C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide via a linker.

[0282] The present disclosure provides a conjugate comprising a CPP comprising the sequence set forth in SEQ ID NO: 13 linked to an antisense oligonucleotide as described herein.

[0283] The present disclosure also provides a conjugate comprising a CPP comprising the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide the nucleotide sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 1.

[0284] The present disclosure also provides a conjugate comprising a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1.

[0285] The present disclosure provides a conjugate consisting of:

[0286] a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13; and

[0287] an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide.

[0288] In one example, the C-terminus of the CPP is linked to the secondary amine at the 3' end of the antisense oligonucleotide. For example, the carboxylic acid C-terminus of the CPP is linked to the secondary amine at the 3' end of the antisense oligonucleotide via a linker.

[0289] The present disclosure further provides a conjugate consisting of:

[0290] a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids;

[0291] an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide.

[0292] Suitable examples of CPPs are described in, e.g, PCT / AU2020 / 051397. In some examples the amino acid sequence of the CPP comprises or consists of: RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 13). In one example, the CPP comprises the sequence RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 13), optionally wherein any amino acid other than glycine is a D amino acid.

[0293] For the avoidance of doubt, a conjugate of the disclosure that “comprises7' (i) a CPP “consisting" of an amino acid sequence and (ii) an antisense oligonucleotide “consisting” of a nucleotide sequence, in some examples, includes an intervening flexible linker comprising or consisting of an amino acid sequence (e.g., SEQ ID NO: 16) distinct from the amino acid sequence of the CPP (SEQ ID NO: 13), that links the CPP to the nucleotide sequence (SEQ ID NO: 1) of the conjugate.

[0294] In some examples, the conjugate consists of: (i) a CPP the amino acid sequence of which consists of the amino acid sequence set forth in SEQ ID NO: 13; (ii) a phosphorodiamidate morpholino oligonucleotide the nucleotide sequence of which consists of the nucleotide sequence set forth in SEQ ID NO: 1; and (iii) a flexible linker linking (i) and (ii) the amino acid sequence of which consists of an amino acid sequence selected from among: GGGGS, GGGGSGGGGS (SEQ ID NO: 14), GAS, GGG, GSG, GTG, GGTAGSTGG (SEQ ID NO: 15), GASGGASG (SEQ ID NO: 16) and GASG. In some preferred examples, the amino acid sequence of the flexible linker consists of the amino acid sequence set forth in SEQ ID NO:16.

[0295] In some examples the conjugate comprises the structure corresponding to formula I as shown in Figure 11.

[0296] In some examples, the conjugate comprises the structure depicted in Figure 13, also referred to as formula “PPMQ10”.

[0297] Pharmaceutical Compositions

[0298] Also provided herein are pharmaceutical compositions comprising any of the foregoing conjugates disclosed herein, and formulated with at least a pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and / or diluent and their use in a method disclosed herein.

[0299] Pharmaceutical compositions containing any of the ASOs described herein, for use in the methods disclosed herein, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In some examples, a pharmaceutical composition for treating a subject comprises a therapeutically effective amount of any ASO disclosed herein.

[0300] Hereinafter, the phrases ‘‘physiologically acceptable excipient” and “pharmaceutically acceptable excipient” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0301] Pharmaceutically acceptable excipients can include, but are not limited to, physiological saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti- oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.. Easton, Pa., latest edition, which is incorporated herein by reference.

[0302] In various examples, pharmaceutical compositions disclosed herein may comprise additional excipients selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like.

[0303] In various examples, the conjugates or pharmaceutical compositions herein are formulated for systemic administration. Suitable routes of administration may, for example, include parenteral delivery, including intramuscular, subcutaneous, intravenous or intraarterial injection. In various examples, the coOnjugates or pharmaceutical compositions are formulated for intravenous administration.

[0304] As the skilled person will be aware, a conjugate or pharmaceutical composition can be administered in a local or systemic manner. Local administration includes injection of the conjugate or pharmaceutical composition directly into a tissue region of a patient to be treated, e.g., directly to the kidney of a subject.

[0305] In some examples, a conjugate or pharmaceutical composition disclosed herein is administered systemically, e g., parenterally, e.g., by intravenous injection.

[0306] In some examples, a conjugate or pharmaceutical composition is administered by injection.

[0307] For injection, the conjugate of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0308] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e g., in ampoules or in multidose containers with optionally, an added preservative.

[0309] Pharmaceutical compositions for parenteral administration include aqueous solutions of the conjugate in water-soluble form. Additionally, suspensions of the conjugate may be prepared as appropriate oily or water-based injection suspensions.

[0310] Alternatively, the conjugate may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0311] Conjugates and pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the conjugate is contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of conjugate (i.e., those disclosed herein) effective to prevent, slow, alleviate, or ameliorate symptoms of a condition, e.g., ADPKD or to reverse a symptom of a condition, e.g., ADPKD.

[0312] In some examples, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 0.3 mg / kg-5.0 mg / kg, e.g., 1.0-2.3 mg / kg to a subject.

[0313] In some examples, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 0.4 mg / kg-5.0 mg / kg, e.g., 1.2 to 4 mg / kg or 1.2-2.4 mg / kg or 0.4 to 1.2 mg / kg to a subject.

[0314] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.0 mg / kg to a subject.

[0315] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.1 mg / kg to a subject.

[0316] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.2 mg / kg to a subject.

[0317] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.3 mg / kg to a subject.

[0318] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.4 mg / kg to a subject.

[0319] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.5 mg / kg to a subject.

[0320] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.6 mg / kg to a subject.

[0321] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.7 mg / kg to a subject.

[0322] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.8 mg / kg to a subject.

[0323] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 1.9 mg / kg to a subject.

[0324] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 2.0 mg / kg to a subject.

[0325] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 2.1 mg / kg to a subject.

[0326] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 2.2 mg / kg to a subject.

[0327] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 2.3 mg / kg to a subject. For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 2.4 mg / kg to a subject.

[0328] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 3 mg / kg to a subject.

[0329] For example, a conjugate of the disclosure is contained in a composition in an amount effective to deliver 4 mg / kg to a subject.

[0330] The disclosure also provides a pharmaceutical composition comprising a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a CPP, wherein the pH of the composition is between 5 and 7.5.

[0331] The disclosure also provides a pharmaceutical composition comprising a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a CPP, wherein the pH of the composition is between 5.0 and 7.0.

[0332] The disclosure also provides a pharmaceutical composition comprising a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a CPP, wherein the pH of the composition is between 5.5 and 6.5.

[0333] The disclosure also provides a pharmaceutical composition comprising a conjugate described herein wherein the pH of the composition is between 5.5 and 7.0.

[0334] In one example, the pH of the pharmaceutical composition is between 5.5 and 6.5. For example, the pH of the pharmaceutical composition is 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the pH of the pharmaceutical composition is 6.0.

[0335] The disclosure also provides a pharmaceutical composition comprising an ASO which binds to a miR-17 binding site in a 3’UTR of aPKDl gene transcript in a cell and a buffer.

[0336] In one example, the ASO comprises a backbone modification. For example, the backbone modification is a phosphorothioate linkage or a phosphorodiamidate linkage or a phosphorodiamidate morpholino linkage. For example, the ASO is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0337] The disclosure also provides a pharmaceutical composition comprising:

[0338] (i) a conjugate of an ASO which binds to a miR-17 binding site in a 3' UTR of a PKD1 gene transcript in a cell and a CPP; and

[0339] (ii) a buffer.

[0340] The disclosure also provides a pharmaceutical composition comprising a conjugate described herein and a buffer.

[0341] In one example, the buffer is a physiological buffer.

[0342] In one example, the buffer is effective to maintain the pH of the composition at a pH of between 5.5 and 7.0. In one example, the buffer is effective to maintain the pH of the composition at a pH of between 5.5 and 6.5. For example, the buffer is effective to maintain the pH of the composition at a pH of 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the buffer is effective to maintain the pH of the composition at a pH of 6.0. Reference herein to a pH will be understood to mean the target pH or the pH in the middle of an acceptable range of pHs, e.g., as indicated in a release criterion for a drug. For example, a pH of 6.0 encompasses a pH of 6.0±0.2 or 0.3.

[0343] In one example, the pH of the pharmaceutical composition is between 5.5 and 6.5. For example, the pH of the pharmaceutical composition is 5.6 or 5.7 or 5.8 or 5.9 or 6.0 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5. For example, the pH of the pharmaceutical composition is 6.

[0344] In one example, the buffer is a phosphate buffer.

[0345] In one example, the pharmaceutical composition additionally comprises a tonicity modifier.

[0346] In one example, the tonicity modifier is sodium chloride.

[0347] The disclosure provides a pharmaceutical composition comprising:

[0348] (i) a conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to an antisense oligonucleotide comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the antisense oligonucleotide by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16, wherein the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0349] (li) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0. In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0350] The disclosure additionally provides a pharmaceutical composition comprising:

[0351] (A) a conjugate consisting of:

[0352] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0353] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0354] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide; and

[0355] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0356] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the antisense oligonucleotide.

[0357] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0358] The disclosure additionally provides a pharmaceutical composition comprising: (A) a conjugate consisting of:

[0359] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0360] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0361] (iii) an antisense oligonucleotide the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the antisense oligonucleotide;

[0362] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0363] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0364] The disclosure provides a pharmaceutical composition comprising:

[0365] (i) a conjugate comprises a CPP the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 13, linked to a PMO comprising or consisting of the sequence set forth in SEQ ID NO: 1, wherein the CPP is linked to the PMO by a linker the amino acid sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 16, wherein the CPP and the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0366] (ii) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0. In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0367] The disclosure additionally provides a pharmaceutical composition comprising:

[0368] (A) a conjugate consisting of:

[0369] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0370] (ii) a linker the amino acid sequence of w hich consists of the sequence set forth in SEQ ID NO: 16, wherein the linker includes only D-amino acids (other than glycine for which there is no D amino acid); and

[0371] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the CPP is linked to the 3' end of the PMO; and (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0372] In one example, the C-terminus of the CPP is linked to the N-terminus of the linker and C-terminus of the linker is linked to the secondary amine at the 3' end of the PMO. In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0373] The disclosure additionally provides a pharmaceutical composition comprising:

[0374] (A) a conjugate consisting of:

[0375] (i) a CPP the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13, wherein the amino acid sequence of the CPP includes only D-amino acids (other than glycine for which there is no D amino acid);

[0376] (ii) a linker the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 16, wherein the amino acid sequence of the linker includes only D-amino acids (other than glycine for which there is no D amino acid) and wherein the N terminus of the linker is linked to the C terminus of the CPP; and

[0377] (iii) a PMO the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the linker is linked to the 3' end of the PMO;

[0378] (B) a phosphate buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0379] In one example, the pharmaceutical composition additionally comprises sodium chloride.

[0380] In one example, the pharmaceutical composition is lyophilized.

[0381] In one example, the lyophilized composition has:

[0382] (i) the conjugate at a purity of at least 90%. as determined by high performance liquid chromatography (HPLC);

[0383] (ii) a total impurity content of less than 10%, as determined by HPLC; and / or

[0384] (iii)a water content of less than 5%, as determined by Coulometric Karl Fischer (CKF) assay.

[0385] In one example, the purity of the conjugate is at least 91%. In one example, the purity of the conjugate is at least 91.5%.

[0386] In one example, the impurity content is less than 9%. In one example, the impurity content is less than 8.6%.

[0387] In one example, the total impurity content, purity and / or water content of the conjugate is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 12 months, or at least 18 months at a temperature of: -20°C ± 5°C and / or 5°C ± 3°C.

[0388] In one example, the composition is a liquid formulation.

[0389] In one example, the composition is or was previously reconstituted from a lyophilized formulation having:

[0390] (i) the conjugate at a purity of at least 90%. as determined by high performance liquid chromatography (HPLC);

[0391] (ii) a total impurity content of less than 10%, as determined by HPLC; and / or (iii)a water content of less than 5%, as determined by Coulometric Karl Fischer (CKF) assay.

[0392] In one example, the purity of the conjugate is at least 91%. In one example, the purity of the conjugate is at least 91.5%.

[0393] In one example, the impurity content is less than 9%. In one example, the impurity content is less than 8.6%.

[0394] In one example, the total impurity content, purity and / or water content of the conjugate is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 12 months, or at least 18 months at a temperature of: -20°C ± 5°C and / or 5°C ± 3°C.

[0395] In one example, the composition is a liquid or reconstituted composition and has: (i) a total impurity content of no more than 7%, as determined by Ultra Performance Liquid Chromatography (UPLC);

[0396] (ii)the conjugate at a purity of at least 92%, as determined by UPLC; and / or

[0397] (iii) an osmolality in the range of 300 mOsm / kg to 310 mOsm / kg.

[0398] In one example, the total impurity content is no more than 6.5%. In one example, the total impurity content is no more than 6.1%.

[0399] In one example, the purity of the conjugate is at least 93.0%. In one example, the purity of the conjugate is at least 93.9%.

[0400] In one example, the osmolality is 303 mOsm / kg or 306 mOsm / kg. Reference herein to an osmolality’ will be understood to mean the target osmolality or the osmolality in the middle of an acceptable range of osmolalities, e.g., as indicated in a release criterion for a drug. For example, an osmolality is 303 mOsm / kg encompasses an osmolality of 303±3 or 2.

[0401] In one example, the total impurities, purity and / or osmolality is determined after storage for a period of at least 1 month, at least 3 months, at least 6 months, at least 9 months, or at least 12 months at a temperature of 5°C ± 3°C.

[0402] In one example, the pharmaceutical composition comprises the conjugate at a purity of at least 90%, as determined by HPLC following incubation at 50°C for three days.

[0403] In one example, the pharmaceutical composition comprises the conjugate at a purity of at least 88%, as determined by HPLC following incubation at 50°C for six days.

[0404] In one example, the pharmaceutical composition is a liquid composition or reconstituted composition comprising the conjugate at a concentration of 20 mg / mL.

[0405] The disclosure also provides a container comprising a conjugate or pharmaceutical composition disclosed herein.

[0406] In one example, the container is a vial and / or a syringe.

[0407] For example, the composition is supplied in a glass vial. In one example, the vial comprises the composition having a volume in the range of 5 mL to 15 mL. For example, the composition has a volume in the range of 7 mL to 12 mL. For example, the formulation has a volume of 10 mL.

[0408] In one example, the composition comprises the conjugate at a concentration of 10 mg / mL.

[0409] The present disclosure additionally provides a container comprising an amount of a conjugate disclosed herein, wherein the container comprises sufficient conjugate for administration at a dose of 10 mg / mL to 30 mg / mL.

[0410] In one example, the conjugate is lyophilized.

[0411] In one example, the container comprises a liquid or reconstituted formulation comprising the conjugate.

[0412] Methods of Treating Conditions

[0413] The present disclosure provides, for example, a method of treating, preventing and / or delaying progression of a condition, e.g., ADPKD. The methods described herein include a method for treating, preventing and / or delaying progression of a condition in a subject in need thereof by administering to the subject a therapeutically effective amount of a conjugate of the present disclosure or a pharmaceutical composition comprising the conjugate disclosed herein. Likewise, in some examples, the conjugate is used in the manufacture of a medicament for treating and / or delaying progression of a condition, e.g., ADPKD.

[0414] In one example, the subject to be treated is suffering from ADPKD. For example, the subject has been diagnosed as having or suffering from ADPKD. For example, the subject is in need of treatment. Such subjects can be administered the conjugate as described here to treat a symptom of ADPKD.

[0415] In one example, administration of the conjugate or pharmaceutical composition as described herein slows progression of a condition, such as ADPKD.

[0416] In one example, administration of the conjugate or pharmaceutical composition as described herein reduces the height adjusted kidney volume of a subject.

[0417] In one example, administration of the conjugate or pharmaceutical composition as described herein reduces the number of cysts detectable in a kidney of a subject.

[0418] In some examples target engagement of the conjugate in kidney cells of the subject is at least about 20% to at least about 80%. In some examples target engagement of the conjugate in kidney cells of the subject is at least 25% to at least 75%. In some examples target engagement of the conjugate in kidney cells of the subject is at least 30% to at least 70%.

[0419] In some examples target engagement of the conjugate in kidney cells of the subject is at least about 38% to at least about 65%. e.g., 40%, 42%, 45%, 50%, 53%, 55%, 60%, 62%, or another target engagement level from about 38% to at least about 65%.

[0420] In one example, administration of the conjugate or pharmaceutical composition as described herein reduces the number of new cysts developing in a kidney of a subject. In one example, administration of the conjugate or pharmaceutical composition as described herein reduces the size of cysts in a kidney of a subject.

[0421] Also provided herein is a method for increasing the level of a transcript, e.g.. PKD1 transcript or PCI protein in a cell, the method comprising contacting the cell with a conjugate or pharmaceutical composition as disclosed herein, whereby the amount of PKD1 transcript or PCI protein in the cell is increased. In on example, the cell is within a subject. In one example, the conjugate or pharmaceutical composition is administered systemically, e.g., intravenously to the subject to thereby contact the cell.

[0422] In some examples, administration to a subject or contact with cells with the conjugate or pharmaceutical compositions disclosed herein increases the level of PCI protein about 1.1 fold to 2 fold, e.g., 1.1 fold or 1.2 fold or 1.3 fold or 1.4 fold or 1.5 fold or 1.6 fold or 1.7 fold or 1.8 fold or 1.9 fold or 2.0 fold compared to the level prior to the administration or contact.

[0423] Suitable routes of administration for treatment with the conjugates, pharmaceutical compositions, or medicaments disclosed herein are disclosed herein and include parenteral routs of administration such as intravenous.

[0424] In one example, the conjugate is administered at a weight-based dose. The term ■‘weight-based dose” means the dose administered is varied based on weight of a subject and, optionally, severity of disease.

[0425] In some examples, the conjugate is administered at a dose of 0.3mg / kg-5.0 mg / kg, e.g.. 1.0-2.3 mg / kg.

[0426] In some examples, the conjugate is administered at a dose of 0.4 mg / kg-5.0 mg / kg, e.g., 0.4-1.2 mg / kg, or 1.2-2.4 mg / kg.

[0427] For example, the conjugate is administered at a dose of 0.4 mg / kg.

[0428] For example, the conjugate is administered at a dose of 0.8 mg / kg.

[0429] For example, the conjugate is administered at a dose of 1.0 mg / kg.

[0430] For example, the conjugate is administered at a dose of 1.1 mg / kg.

[0431] For example, the conjugate is administered at a dose of 1.2 mg / kg.

[0432] For example, the conjugate is administered at a dose of 1.3 mg / kg.

[0433] For example, the conjugate is administered at a dose of 1.4 mg / kg.

[0434] For example, the conjugate is administered at a dose of 1.5 mg / kg.

[0435] For example, the conjugate is administered at a dose of 1.6 mg / kg.

[0436] For example, the conjugate is administered at a dose of 1.7 mg / kg.

[0437] For example, the conjugate is administered at a dose of 1.8 mg / kg.

[0438] For example, the conjugate is administered at a dose of 1.9 mg / kg.

[0439] For example, the conjugate is administered at a dose of 2.0 mg / kg.

[0440] For example, the conjugate is administered at a dose of 2.1 mg / kg.

[0441] For example, the conjugate is administered at a dose of 2.2 mg / kg.

[0442] For example, the conjugate is administered at a dose of 2.3 mg / kg. For example, the conjugate is administered at a dose of 2.4 mg / kg.

[0443] For example, the conjugate is administered at a dose of 3.0 mg / kg.

[0444] For example, the conjugate is administered at a dose of 3.5 mg / kg.

[0445] For example, the conjugate is administered at a dose of 3.5 mg / kg.

[0446] For example, the conjugate is administered at a dose of 4.0 mg / kg.

[0447] In some examples, the conjugate is administered multiple times to the subject.

[0448] In some examples, there is between 2 weeks and 4 months between each dose.

[0449] In some examples, there is 4 weeks between each dose.

[0450] In some examples, there is 6 weeks between each dose.

[0451] In some examples, there is 8 weeks between each dose.

[0452] In some examples, there is 12 weeks between each dose.

[0453] In some examples, there is 1 month between each dose.

[0454] In some examples, there is 2 months between each dose.

[0455] In some examples, there is 3 months between each dose.

[0456] Medical products

[0457] The present disclosure also provides an article of manufacture, e.g., a container comprising a conjugate or pharmaceutical composition.

[0458] For example, the container is a vial.

[0459] For example, the container is a prefilled glass vial.

[0460] In one example, the container comprises sufficient conjugate for administration at a dose between 0.3 mg / kg-5.0 mg / kg or 0.5 mg / kg-5.0 mg / kg, e.g., 1.0-2.3 mg / kg.

[0461] In one example, the container comprises sufficient conjugate for administration at a dose between 0.4 mg / kg-5.0 mg / kg or 0.4 mg / kg-2.4 mg / kg, e.g., 0.4-1.2 mg / kg, or 1.2-2.4 mg / kg.

[0462] For example, the container comprises sufficient conjugate for administration at a dose of 0.4 mg / kg.

[0463] For example, the container comprises sufficient conjugate for administration at a dose of 0.8 mg / kg.

[0464] For example, the container comprises sufficient conjugate for administration at a dose of 1.0 mg / kg.

[0465] For example, the container comprises sufficient conjugate for administration at a dose of 1.1 mg / kg.

[0466] For example, the container comprises sufficient conjugate for administration at a dose of 1.2 mg / kg.

[0467] For example, the container comprises sufficient conjugate for administration at a dose of 1.3 mg / kg. For example, the container comprises sufficient conjugate for administration at a dose of 1.4 mg / kg.

[0468] For example, the container comprises sufficient conjugate for administration at a dose of 1.5 mg / kg.

[0469] For example, the container comprises sufficient conjugate for administration at a dose of 1.6 mg / kg.

[0470] For example, the container comprises sufficient conjugate for administration at a dose of 1.7 mg / kg.

[0471] For example, the container comprises sufficient conjugate for administration at a dose of 1.8 mg / kg.

[0472] For example, the container comprises sufficient conjugate for administration at a dose of 1.9 mg / kg.

[0473] For example, the container comprises sufficient conjugate for administration at a dose of 2.0 mg / kg.

[0474] For example, the container comprises sufficient conjugate for administration at a dose of 2.1 mg / kg.

[0475] For example, the container comprises sufficient conjugate for administration at a dose of 2.2 mg / kg.

[0476] For example, the container comprises sufficient conjugate for administration at a dose of 2.3 mg / kg.

[0477] For example, the container comprises sufficient conjugate for administration at a dose of 2.4 mg / kg.

[0478] For example, the container comprises sufficient conjugate for administration at a dose of 2.7 mg / kg.

[0479] For example, the container comprises sufficient conjugate for administration at a dose of 3.0 mg / kg.

[0480] For example, the container comprises sufficient conjugate for administration at a dose of 3.5 mg / kg.

[0481] For example, the container comprises sufficient conjugate for administration at a dose of 4.0 mg / kg.

[0482] For example, the container comprises sufficient conjugate for administration at a dose of 4.3 mg / kg.

[0483] For example, the container comprises sufficient conjugate for administration at a dose of 5 mg / kg.

[0484] In the discussion above, reference to “a dose sufficient” will be understood that the container comprises more than the recited amount to account for different weights of subjects and the component of conjugate that is not or cannot be removed from the container during dosing. The conjugate can be in dried, lyophilized or liquid form.

[0485] The present disclosure is not to be limited by the following examples.

[0486] EXAMPLES

[0487] Example 1. PPMO localisation in the kidneys of non-human primates from a dose range finding study

[0488] A PMO with sequences corresponding to PKD1 H463UTR(+604+628) (SEQ ID NO: 1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) with an intervening linker to generate peptide-PMOs (PPMOs). Vehicle control or PPMO at 3, 10, 15 (a PPMO without an intervening amino acid linker) or 30 mg / kg were administered to the non-human primates via intravenous infusion. Organs were collected for analysis 28 days post-treatment, fixed in 10% formalin for 24 hours, and processed for paraffin embedding. miRNAScope assays were conducted using an ACD Bio miRNAScope kit, which included hybridization with a probe specific to the PPMO, followed by signal amplification and visualization. Images were captured wi th a 3DHISTECH Pannoramic Flash 250 III Slide Scanner at 20x magnification. The PPMO staining appears in dark grey, while hematoxylin counter-staining highlighted kidney morphology in light grey. The figure presents representative images of transverse kidney sections from an animal treated with vehicle control (Figure 1A) and another treated with 30 mg / kg PPMO (Figure IB). Additionally, zoomed-in images of three regions each in the cortex and the medulla are depicted. Animals that received the vehicle control displayed no drug signal, while those treated with the drug exhibited a strong signal indicating the presence of PPMO in the kidney medulla and cortex. Within those kidney structures, the PPMO distributes to proximal and distal convoluted tubule cells (cortex) and tubule cells (medulla).

[0489] Example 2. Semi-quantitative analysis of PPMO signal in the kidney of non-human primates from a dose range finding study

[0490] Images from all animals described in Example 1 were further analysed using a semi-quantitative automated histological scoring method to determine the optical density (OD) of probe staining in both the kidney cortex and medulla (Figure 2 A). The OD of images from treated animals was normalized the OD of images from vehicle treated animals. The ratio of OD in images from cortex versus medulla of treated animals is depicted in Figure 2B. The data in both graphs are represented as geometric mean + / - standard error of the mean (n=2). PPMOAcontains the same CPP and PMO (SEQ ID NOs: 1 (+604+628)) as PPMO but uses an alternative linker chemistry to combine the moieties. PPMO and PPMOAdistribute to the cortex and medulla in NHP kidneys. The OD signal was generally higher in the cortex than the medulla, with the 10 mg / kg dose having the largest differential between cortex and medulla. Example 3. PPMO tissues concentration in non-human primates from a dose range finding study

[0491] A PMO with sequences corresponding to PKD1 H463UTR(+604+628) (SEQ ID NO: 1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). Vehicle control or PPMO at 3, 10 or 30 mg / kg were administered to the non-human primates via intravenous infusion. Kidney, liver, pancreas, heart, and skeletal muscle were collected for selected doses and time points. PPMO amounts in the tissue lysates were measured using a qualified ELISA method and normalized according to the amount of tissue processed to calculate the PPMO tissue concentration. Drug concentrations in the kidney were assessed 48 hours (for the 3 mg / kg group only) and 28 days post-treatment (Figure 3A). Additionally, drug concentrations in the kidney, liver, pancreas, heart, and skeletal muscle were analysed after administering 3 mg / kg PPMO at designated time points (Figure 3B). At Day 3 and Day 29, mean tissue concentrations of PPMO were high in the kidney and in the liver, with preferential distribution to the kidney. Concentrations in heart, skeletal muscle, and pancreas were low.

[0492] Example 4. PPMO concentration in tissues in non-human primates from a pharmacokinetics study

[0493] A PMO with sequences corresponding to PKD1 H463UTR(+604+628) (SEQ ID NO: 1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). Animals were intravenously infused with PPMO at doses of 10 or 30 mg / kg. Tissues including the kidney cortex, kidney medulla, liver, pancreas, and skeletal muscle were collected at specific doses and time points. The concentrations of PPMO in the tissue lysates were measured using a qualified ELISA method and normalized according to the amount of tissue processed, enabling the calculation of PPMO tissue concentration. The drug concentrations in kidney cortex and kidney medulla samples were evaluated 24 hours posttreatment for all groups and at 56 days post treatment for the 10 mg / kg group (Figure 4 A). Additionally, the drug concentrations in the kidney cortex, kidney medulla, liver, pancreas, and skeletal muscle were measured at specified time points following dosing with 10 mg / kg PPMO. The mean tissue concentrations of PPMO were high in the kidney cortex, kidney medulla and in the liver, with preferential distribution to the kidney cortex and liver. Concentrations in skeletal muscle and pancreas were low;

[0494] Example 5. PPMO effect on PCI protein levels in kidneys of non-human primates from a pharmacokinetics study

[0495] Selected samples from animals described in Example 4 were further analysed to assess the PPMO effect on PCI protein levels. The level of PCI in kidney medulla samples from animals treated with vehicle or PPMO at 10 mg / kg was assessed using western blot assay. Protein was extracted form the day-28 post treatment samples using RIPA buffer supplemented with protease inhibitor cocktail and 2x PhosSTOP. Protein lysates were generated using a homogeniser. Samples were cleared, total protein quantitated using BCA protein kit and run on protein gels. The protein was transferred to nitrocellulose membrane by wet transfer. The membrane was stained for total protein and mouse anti-PCl (Santa-Cruz, cat no scl30554) primary’ antibody followed by anti-mouse (IRDye® 800CW preabsorbed) secondary antibody. Blots were imaged on an Odyssey Imager, quantitative analysis was performed using Image Studio Ver 5.5 software. The raw fluorescence signal for PCI was first normalised to the raw fluorescence signal of loading control (total protein) and then expressed as fold-change relative to samples from vehicle treated animals. The data of n=2 western blot assays is presented as mean + / - standard error of the mean. The kidney medulla samples PPMO treatment at 10 mg / kg increases PC 1 protein by 1.27 -fold compared to vehicle treatment (Figure 5).

[0496] Example 6. PPMO localisation the in kidneys of normal and Pkdl deficient mice PPMO distribution in healthy and cystic kidneys was assessed utilising a tamoxifen-inducible conditional knock-out (KO) mouse model of Pkdl. Mice without tamoxifen induction are healthy, while treatment with tamoxifen will result in deletion of the Pkdl gene and consequently development of a ADPKD phenotype. At the age of PND70 healthy and diseased mice were treated with 10 mg / kg of a PMO with sequences corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO:1) conjugated to a cell penetrating peptide (SEQ ID NO:47). Kidneys were collected for analysis 3 days post-treatment, fixed in 10% formalin for 24 hours, and processed for paraffin embedding. miRNAScope assays were conducted using an ACD Bio miRNAScope kit. which included hybridization with a probe specific to the PPMO, followed by signal amplification and visualization. Images were captured with a 3DHISTECH Pannoramic Flash 250 III Slide Scanner at 20x magnification (Figure 6). The PPMO staining appears in dark grey, while hematoxylin counter-staining highlighted kidney morphology in light grey. Additionally, zoomed-in images of three regions each in the cortex and the medulla are depicted. Healthy animals exhibit normal kidney histopathology, with PPMO detected in the kidney medulla and cortex. In Pkdl KO animals, characteristic cysts appear in both the medulla and cortex, with PPMO staining present in the cyst lining cells and non-cystic cells.

[0497] Example 7. Analysis of PPMO target engagement at PKD1 mRNA and corresponding PCI increase.

[0498] A PMO with sequences corresponding to PKD1 H463UTR(+604+628) (SEQ ID NO: 1) was conjugated to a cell penetrating peptide (SEQ ID NO: 13) to generate peptide-PMOs (PPMOs). The PPMO was tested across multiple cell systems (as listed below) to examine the relationship between target engagement, defined as binding of the PMO component to the 3’ UTR of PKD1 mRNA, and upregulation of PCI protein.

[0499] Panel A in each of Figures 7-9 shows target engagement data assessed by digital droplet PCR (ddPCR). measuring inhibition of reverse transcription (RT) caused by PMO binding to the PKD1 3' UTR. Cells were treated with various doses of PPMO for 48-72 hours, after which total RNA was extracted using the MagMAX Total RNA 96 kit. Expression of PKD1 was quantified using TaqMan ddPCR (probe: Hs00947394_gl ). normalized to the housekeeping gene DHX57 (probe: Hs00376574_ml), and expressed as fold-change relative to untreated cells. Anon-targeting control oligonucleotide (GTC CTR), which does not hybridize to human transcripts, was included as a negative control.

[0500] Panel B presents the corresponding PCI protein expression data from the same cell models. Cells were incubated with PPMO or control for either 72 hours or 5 days. Protein lysates were collected at day 5 using RIPA buffer with 2% protease inhibitor cocktail and 2x PhosSTOP. Protein concentrations were determined using a BCA assay, and samples were separated on NuPAGE 3-8% Tris-Acetate gels. Proteins were transferred to nitrocellulose membranes by wet transfer, stained for total protein, and probed with a mouse anti-PCl antibody (Santa Cruz, cat. no. scl30554) followed by an IRDye® 800CW-labeled anti-mouse secondary antibody. Imaging was performed with an Odyssey Imager, and quantification was done using Image Studio v5.5. PCI fluorescence signal was normalized to total protein signal and reported as fold-change versus untreated control. A positive control oligonucleotide, RGLS4326 (Med Chem Express, cat. no. HY-139290). an inhibitor of miR-17, was included for assay validation.

[0501] Figure 7: In HEK293 cells, treatment with 60 pM PPMO led to 54% target engagement and a 1.75-fold increase in PCI protein level at day 3.

[0502] Figure 8: In an ADPKD patient-derived cell line (from a proximal tubule cyst; PKD1 mutation p. Q2556*), 60 pM PPMO treatment resulted in 53% target engagement and a 1.2-fold increase in PCI protein after 5 days.

[0503] Figure 9: In an iPSC line reprogrammed from PBMCs of an ADPKD patient (PKD1 mutation p. A1458fs), 30 pM PPMO led to 35% target engagement and a 1.5-fold increase in PCI protein at day 5.

[0504] Example 8. PPMO-mediated target engagement in a 3D cyst model derived from ADPKD patient kidneys

[0505] Target engagement in a patient-derived 3D cyst model was assessed using peptide-PMOs (PPMOs), generated by conjugating a PMO with sequences corresponding to PKD1 H46 3UTR(+604+628) (SEQ ID NO:1) to a cell penetrating peptide (SEQ ID NO: 13). The model utilizes cystic epithelial cells isolated from kidneys donated by ADPKD patients. When cultured ex vivo in a 3D matrix, these cells spontaneously form cysts, allowing evaluation of drug effects on cyst growth. Immediately after seeding, cells were co-treated with PPMOs dissolved in molecular-grade H2O at a concentration of 20 pM. After 6 days, total RNA was extracted using the MagMAX Total RNA 96 kit. PKD1 expression was quantified by TaqMan ddPCR (probe: Hs00947394_gl). normalized to DHX57 (probe: Hs00376574_ml), and expressed as fold-change relative to untreated controls. A non-targeting control oligonucleotide (GTC CTR). with no complementarity to human transcripts, served as a negative control. Across two ADPKD donor lines (huPKD04 and huPKDll), target engagement ranged from 38% to 65% (Figure 10).

[0506] Example 9. Formula I conjugate drug product formulation and specification

[0507] In one example, the Formula I drug product (DP) is a clear, colourless solution for intravenous (IV) infusion. The product is supplied at a concentration of 20 mg / mL in a Type III Borosilicate 10R Glass vial with elastomeric stopper and plastic-aluminium seal, all compliant to United States Pharmacopeia (USP) <1207>.

[0508] A single use vial of Formula I DP contains 200 mg of Formula I conjugate in 10 mL of vehicle for IV infusion use. The composition of Formula I drug product is summarised in Table 1. Amounts of excipients may vary as this DP formulation is not final, and adjustments will be made based on concentration of the active pharmaceutical ingredient (API). No other excipients will be included.

[0509]

[0510] Abbreviations: API = Active pharmaceutical ingredient; q.s = quantum satis', USP = United States Pharmacopeia

[0511] Example 10. 56-Day Non-GLP Biodistribution and Toxicokinetics of Formula I in Monkeys

[0512] In the following non-GLP study, the distribution and toxicokinetics of Formula I in monkeys were both evaluated over 56 days post a single dose of Formula I when administered by IV infusion. As part of this study, animals were evaluated for viability (morbidity / mortality), clinical observations, and gross (necropsy) evaluation. For the biodistribution and TK portion of this study male Cynomolgus monkeys were assigned into 1 vehicle control group and 4 treatment groups of 3 monkeys in control group, 12 monkeys in the low dose group, 4 monkeys in the mid dose group and 2 and 3 monkeys in the high dose groups, respectively. The animals received vehicle control (PBS), or Formula I formulated in vehicle at a dose of 10 mg / kg (low dose), 30 mg / kg (mid dose) or 45 mg / kg and 75 mg / kg (high dose groups) by a 60-minute IV infusion.

[0513] Blood samples were collected from all surviving animals in the treatment groups from 10 and 30 mg / kg groups at predose, at the end of infusion (0) and at 0.5, 1, 2, 6, 24, 72, 168, 336, 672 and 1344 (10 mg / kg only) hours post the end of infusion on study Day 1, from the 45 and 75 mg / kg groups at pre-dose, 0, 0.5, 1, 2, 4 (one animal in the 75 mg / kg group only) and 6 hours post-dose, and from the vehicle control group predose and at 192 and 672 hours post the end of infusion on study Day 1. Plasma was obtained within 2 hours of blood collection and stored at <-60°C until analysed.

[0514] Main organs were collected for bioanalysis including the kidney (cortex and medulla separated), brain (cerebellum and frontal lobe), pancreas, skeletal muscle (left tibialis posterior and right biceps brachii), heart, nerve, skin and liver. Tissues were snap-frozen in liquid nitrogen and stored at <-60°C until analysed.

[0515] The quantitation of Formula 1 concentrations in plasma was performed using a qualified ELISA method and the quantitation of Formula I in tissue was conducted using a qualified ELISA method.

[0516] Toxicokinetic Results

[0517] Following a single 60-minute IV infusion administration of Formula I at 30 mg / kg and 10 mg / kg in male Cynomolgus monkeys, Formula I showed a plasma clearance (Cl) of 1.13 ± 0.158 mL / min / kg and 2.33 ± 0.634 mL / min / kg, respectively. The volume of distribution at steady-state (Vdss) was 4400 mL / kg and 14500 ± 4390 mL / kg, respectively. The area under the plasma concentration-time curve from time zero extrapolated to infinity post dosing (AUCO-inf) values were 451000 ± 70200 ng h / rnL and 77500 ± 28500 ng h / mL, respectively. The systemic exposure (Cmax and AUCO-inf) of Formula I increased in a dose-proportional manner from 10 mg / kg to 30 mg / kg. TK parameters following IV infusion of Formula I at 10 and 30 mg / kg to monkeys are shown in Table 2. Table 2 - Toxicokinetics of Formula I in Monkeys following Single IV Infusion

[0518]

[0519] non applicable

[0520] Notes for Table 3: ND: Not determined. The SD values were not determined due to there being less than 3 values for calculation

[0521] a: Mean values of Vdsspost-intravenous infusion at 30 mg / kg were calculated with PK profiles from two animal samples up to 672 hours, which indicated a more precise evaluation on Vdss- The PK results up to 672 hours showed 2-phase decay, indicating more complete PK profiles compared to those up to 24 hours. Biodistribution Results

[0522] Tissue concentrations of Formula I in the control group were all below the limit of quantification (BLQ). The data indicate that the control group was not exposed to the test article.

[0523] After a single IV infusion of Formula I at 30 mg / kg and 10 mg / kg in male Cynomolgus monkeys, the test article was primarily detected in the kidney cortex, kidney medulla and liver of the animals, generally showing concentrations that were hundreds of times higher than those in other tissues. At 10 mg / kg, the highest tissue exposure (AUCo-iast) was observed in kidney cortex (482000000 h*ng / g), liver (358000000 h*ng / g), kidney medulla (41700000 h*ng / g), and heart (22500000 h*ng / g), followed by substantially lower exposure in pancreas, nerve (sciatic), skin, muscle and brain. The mean concentrations of Formula I in kidney cortex, liver and kidney medulla remained high at 1344 hours (Day 57) at 46500, 13900 and 2380 for the 10 mg / kg dosing group, respectively, while the mean concentrations of Formula I in other tissues were close to or below the lower limits of quantification (LLOQ). In conclusion, IV infusion of Formula I at 10 and 30 mg / kg was well tolerated in monkeys and, similar to mice, resulted in the highest tissue concentration in the kidney (target organ).

[0524] Example 11. Repeat Dose GLP Toxicokinetics of Formula I in Cynomolgous Monkeys The TK profile of Formula I was assessed in a 12-week GLP repeat dose study in monkeys with doses administered via IV infusion every 4 weeks for a total of 4 doses, with the reversibility or persistence of any potential effects assessed after a 12-week recovery period. Cynomolgus monkeys (n=6 / sex in control and high dose groups and n=4 / sex in low and mid dose groups) were administered 0, 4.8, 12, or 30 mg / kg of Formula I once every 4 weeks for a total of 4 doses (on Days 1. 29. 57 and 85). Details of the toxicology study are provided in Example 16. On Days 1 and 85 blood samples were collected pre-dose, at 1-5 minutes. 30 minutes, 1. 2, 4, 8, 24, 48, 96, 168, 336 and 672 hours post end of infusion from all available animals. Plasma was obtained within 2 hours of blood collection and stored at < -60°C until analysed. The quantitation of Formula I concentrations in plasma was performed using a validated ELISA method.

[0525] The toxicokinetic parameters are summarised on the following page in Table 3. Table 3 - Toxicokinetics of Formula I following 12-Week Repeat Dose in Monkeys

[0526]

[0527] Notes: Data is presented as mean ± SD for Cmax and AUC values, and median (range) for Tmax values NA = Not applicable

[0528] After single (Day 1) or repeated (Day 85) IV infusions of Formula I to male and female monkeys, median Tmax values for Formula I were observed at 1.1 hours post start of infusion.

[0529] No marked sex differences in systemic exposure (AUCo-49h and Cmax) to Formula I was observed at any dose level. As the dosage increased from 4.8 to 12 mg / kg / dose, and from 12 to 30 mg / kg / dose, the systemic exposure (AUCo-49h and Cmax) to Formula I increased dose proportionally following administration on Days 1 and 85. Overall, as the dosage increased from 4.8 to 30 mg / kg / dose, the systemic exposure as measured by AUC (AUCo-49h) to Formula I increased more than dose-proportionally following administration on Days 1 and 85. however Cmax increased dose-proportionally. No marked drug accumulation was observed at any dose level.

[0530] Example 12. Metabolic Profiling and Identification of Formula I in Plasma and Urine in Monkeys

[0531] The aim of this study w as to identify the metabolites of Formula I in plasma and urine following IV infusion in Cynomolgus monkeys by LC-UV-HRMS, and to propose the metabolic pathways of Formula I. Monkeys were dosed with Formula I at 30 mg / kg, and plasma and urine samples were pooled at 6- and 48-hours postdose, and on Day 2 post-dose, using equal volumes, respectively. Samples were analysed by LC-UV-HRMS, and the structures of the metabolite were proposed based on the interpretation of the MS and MS2 data.

[0532] Similar to mice, in addition to unchanged Formula I, two metabolites of Formula I were detected in monkey plasma and urine. The metabolite was assigned as below:

[0533] Ml, Hydrolysis metabolite (P + H2O - | Ac| rrsrtaraGrpGmssrpsaprG); M2, Hydrolysis metabolite (P + H2O - [Ac] rrsrtaraGrpGmssrpsaprGasGG), where P is defined as the parent molecule.

[0534] In pooled monkey plasma of 6 h and 48 h post-dose. Formula I and its 2 metabolites (Ml and M2) were detected at both timepoints. Ml and M2 accounted for 0.05% and 0.19% of the total related components, respectively at 6 h, and 3.75% and 23.97% of the total related components of Ml and M2, respectively at 48 h.

[0535] In pooled monkey urine of Day 2 post-dose, Formula I and its 2 metabolites (Ml and M2) were also detected. Ml and M2 accounted for 46.93% and 51.02% of the total related components, respectively.

[0536] Intact Formula I conjugate accounted for 99.76%, 72.29% and 2.05% of the total related components in pooled monkey plasma at 6 h post-dose, pooled monkey plasma at 48 h postdose and pooled monkey urine at Day 2 post-dose, respectively. Based on the identified metabolites, the major metabolic pathway of Formula I in monkey plasma and urine was proposed as hydrolysis of the peptide component of Formula I.

[0537] Example 13. Single Dose GLP Toxicity Study of Formula I in Cynomolgus Monkeys A single dose GLP toxicity study was conducted in Cynomolgus monkeys to evaluate the potential toxicity of Formula I with an observation period of 3- or 29-days to assess the reversibility or persistence of any toxicities observed or observe the occurrence of any delayed potential toxic effects. Cynomolgus monkeys (n=8 / sex / group) were administered 0 (Formula I Injection Placebo). 4.8, 12, or 30 mg / kg Formula I via a single 60-minute IV infusion. The concentration of Formula I in each dose formulation was confirmed to be within 90% to 110% of nominal using a validated method. Blood samples for TK analysis were collected and analysed as described in Example 11.

[0538] The first 4 animals / sex / group were sacrificed on Day 3, and the last 4 animals / sex / group were sacrificed on Day 29. At termination, a gross necropsy was performed, key organs / tissues were weighed and collected for histopathological observations.

[0539] Detailed clinical observations were performed at least once during pretest for all animals, at least once prior to dosing on Day 1 for all main study animals, and at least once on Day 1 (approximately 1 hour post-dose) and at least once weekly on non-dosing days for all animals. Cage side morbidity and mortality observations were performed at least once daily throughout the study. Local tolerance of the injection site was observed pre-dose, 1-3, 24, and 48 hours post-dose for signs of erythema, oedema, or eschar formation. Body weight measurements were performed at least once weekly throughout the study. Food consumption was assessed by daily visual inspections. Ophthalmic examinations were performed at least once pre-dose, and once on Day 3 and once in Week 4. Blood samples for haematology and clinical chemistry were obtained once pre-dose, once on Day 3, Day 8, and once just prior to scheduled necropsy. Urine samples were collected once pre-dose, once on Day 3, Day 8, and once just prior to scheduled necropsy for urinalysis. Blood samples for cytokine (IL-6, CXCL10, IL- 13, TNF-a, IFN-y, IL-8 and MCP-1) analysis were taken from all animals at 2, 8, 24. and 48 hours post-dose (end of infusion). Blood samples for complement (C3a. C5a and Bb) analysis were taken from all animals at 0 (pre-dose) and 0.5 (30 minutes) hours postdose (end of infusion).

[0540] All animals survived until their scheduled necropsy. There were no test article-related changes in body weight / body weight gain, ophthalmologic examinations, plasma complements (C3a, C5a and Bb), levels of IL- ip and TNF-a, IL-8 or INF-y nor any gross observations at necropsy in this study.

[0541] Transient test article-related clinical signs included skin discolouration on the cheek or nose in males at > 12 mg / kg, and localised swelling at > 12 mg / kg in males and females on Days 1 which in some instances persisted until Days 2 or 3. Soft abnormal stool was noted in both sexes at 30 mg / kg on Days 1, 13 or 28. Additionally, two males at 30 mg / kg showed inappetence on Day 1 and / or Day 13, but all incidences of abnormal stool and inappetence recovered by the day following the observation.

[0542] Compared with both concurrent controls and pretest data, apparent test article-related increases in leukocyte counts were noted at 30 mg / kg in males, and absolute numbers and percent lymphocytes and basophils were noted at 30 mg / kg in males and / or females on Day 29. Apparent test article-related increased fibrinogen (FIB) was noted in both sexes at 30 mg / kg on Day 3 only.

[0543] Compared with concurrent controls and pretest data, test article-related changes in chemistry included increased total bilirubin (females only), urea and CRE in both sexes at 30 mg / kg mostly on Day 3. Increases in urea at > 12 mg / kg and CRE 30 mg / kg were also present on Days 8 and 29. Decreased Mg at 30 mg / kg was noted in both sexes on Days 3, 8 and 29, while decreased Cl was noted in females on Day 3 and in males on Days 8 and 29. In females on Day 3, apparent test article-related increases in alanine aminotransferase (ALT) was observed at 30 mg / kg. Increases in aspartate aminotransferase (AST) were also observed in females on Day 3; however, this finding was of an uncertain relationship to the test article.

[0544] Apparent test article-related changes in urinalysis consisted of positive urinary protein in males at 30 mg / kg on Day 3 and in females at 30 mg / kg on Day 3 and Day 8. Other urinalysis findings observed in males at 30 mg / kg with uncertain relationship to the test article consisted of positive urinary glucose, positive urinary leucocytes and positive occult blood. All changes in urinalysis had recovered by Day 29. The observed changes in urinalysis correlated with the changes in serum urea, CRE, Cl and Mg, as well as tubular degeneration indicated by microscopic observations.

[0545] Test article-related increases in IL-6 and MCP-1 were noted at 30 mg / kg at 2 hours postdose, and increases in CXCL10 were noted at 2 hours post-dose in all dose groups in a dose- dependent manner. Levels of IL-6 returned to baseline in most animals by 8 hours post-dose, while MCP-1 and CXCL10 returned to baseline by 48 hours.

[0546] On Day 3, mean kidney weights (absolute, and body and brain weight ratios) were higher in males dosed at 30 mg / kg compared to controls. By Day 29, no Formula I-related alterations in organ weight parameters were observed.

[0547] Adverse microscopic findings were identified in the kidney at 30 mg / kg. Test article-related microscopic findings in the kidneys on Day 3 included minimal to marked tubular degeneration, often accompanied by minimal neutrophilic inflammation, which correlated with increased kidney weights in males at 30 mg / kg and with increased blood urea, blood CRE, and urine protein in both sexes on Day 3; a slight increase in the incidence and / or severity of minimal to moderate mononuclear cell infiltration; and dose-dependent, minimal or mild tubular basophilic granules. At the moderate and marked severity levels, tubular degeneration was considered adverse due to its potential to affect nephron function. Test article-related microscopic findings in the kidneys on Day 29 included minimal or mild tubular regeneration, often with minimal or mild neutrophilic inflammation, which correlated with increased blood urea and blood CRE; minimal or mild fibrosis with minimal or mild tubular atrophy; a slight increase in the incidence and / or severity of minimal to mild mononuclear cell infiltration; and minimal tubular basophilic granules. Fibrosis and tubular atrophy indicate healing of irreparable kidney injury and were also considered adverse.

[0548] In conclusion, a single IV infusion of Formula I administered to monkeys once at 4.8, 12 or 30 mg / kg followed by a 3-day or 29-day observation period was well tolerated at 4.8 and 12 mg / kg but resulted in adverse microscopic changes (renal tubular degeneration) hich was noted at 30 mg / kg with associated clinical pathology findings.

[0549] Under the conditions of the study, the NOAEL was determined to be 12 mg / kg. The sex combined corresponding mean AUCo-673h and Cmax at the NOAEL were 111000 h*ng / mL and 57400 ng / mL, respectively.

[0550] Example 14.28-Day Non-GLP Maximum Tolerated Dose of Formula I in Monkeys The purpose of this study was to determine the maximum tolerated dose (MTD) of Formula I, when administered by single dose IV infusion to monkeys, to assess the reversibility, persistence, or delayed occurrence of toxic effects over a 29-day observation period.

[0551] For the toxicity phase of this study (Phase I) Cynomolgus monkeys (n=l / sex / group) were randomly assigned and received a single dose of either vehicle control (PBS) or Formula I at 3, 10 or 30 mg / kg via a 60-minute IV infusion on Day 1.

[0552] Viability (mortality and moribundity) checks were performed twice daily, except on the days of animal necropsy, when animals were examined once. Detailed observations were conducted once during pretest for all animals, once prior to dosing on Day 1 for all study animals, and once on Day 1 (within 30 minutes post-dose) and once weekly on non-dosing days for all study animals. Detailed observations were also conducted once on the day of scheduled necropsies. Cage side observation was conducted once daily during pretest from Day -3, and twice daily thereafter except for on the dosing day when cage side observations were performed three times (once before and twice after dosing). Body weight and food consumption was measured pretest and throughout dosing. All animals were also evaluated for haematology, clinical chemistry, coagulation, urinalysis, urine chemistry', biomarker [Kim-1, nGAL, Creatinine (BM), Poly cystin-1 (PCI), histamine, cTnl and cTnT], cytokine analysis, bone marrow, organ weight, gross evaluation and histopathological evaluation. At necropsy, organs were collected for gross pathology' and histopathology.

[0553] All animals survived to scheduled necropsy on Day 29. No significant test article related body weight or food consumption changes yvere observed, yvith only inappetence (2 / 2 animals) and abnormal vocalisation (1 / 2 animals) noted at 30 mg / kg Formula I on Day 1.

[0554] No test article-related changes in haematology were reported during this study. For coagulation markers and clinical chemistry only increased FIB and urea values were observed on Day 3 at 30 mg / kg, both of which were fully (FIB) or partially (urea) resolved at the end of the observation period.

[0555] Test article related changes in bone smear included increased monocytes and erythroid cells and decreased myeloid cells and myeloid: erythroid (M: E) ratio in the male at 30 mg / kg. No Formula I related changes in urinalysis, urine chemistry’, urine biomarker and cytokine analysis were reported. Further, no organ weight, or macroscopic and microscopic Formula I related findings yvere reported.

[0556] In conclusion, Formula I when administered once via 60-minute IV infusion to monkeys at doses of 3, 10 or 30 mg / kg was well tolerated. Under the conditions of the study, 30 mg / kg was considered to be the MTD for Formula I.

[0557] Example 15 - 56-Day Non-GLP Toxicity Study of Formula I in Monkeys

[0558] The toxicity of Formula I in monkeys at the doses of 45 and 75 mg / kg yvas evaluated in a non-GLP study, following a single dose of Formula I administered by IV infusion. Other doses were also included in this study to assess pharmacodynamic and pharmacokinetic effects of Formula I in monkeys.

[0559] In this study, male Cynomolgus monkeys were assigned into 5 groups (n=2 to 12 / group). The animals received vehicle control (PBS), or Formula I at 10, 30, 45 or 75 mg / kg formulated in vehicle by a 60-minute IV infusion on Day 1. Blood and tissue samples for TK and biodistribution analysis were collected and analysed from Pre-dose and on Day 1 of administration, up to Day 57, see Example 11 for details.

[0560] Detailed clinical observations were performed at least once during pretest for all animals, prior to dosing on Day 1, at least once after dosing on Day 1 (at approximately l±0.5 hours post-dose) and once weekly thereafter. Detailed observations were also conducted once on the day of scheduled necropsies. Cage side morbidity and mortality observations were performed at least twice daily and cage side observations for general health and appearance were performed at least once daily throughout the study.

[0561] Samples for clinical pathology (haematology, coagulation, clinical chemistry and urine analysis) were collected predose for the animals, in the toxicity portion of the study (control group and 45 and 75 mg / kg dose groups), but due to early termination of dosed animals, were only partly obtained post dosing. Furthermore, for animals in the toxicity portion of the study cytokines (IL-6, TNF-a, IL-10 and CXCL10) for serum analysis were assessed and at necropsy gross observations and organ weight data and bone marrow smears were collected, and a complete list of organs and gross lesions and masses were assessed for histopathology.

[0562] For animals in other dose groups urinary biomarkers (Kim-1, nGAL, CRE) were assessed and at necropsy gross observations were performed. Animals in the dose groups of the toxicity portion of the study (45 and 75 mg / kg) were necropsied based on the clinical observation endpoint and animals in the control and remaining treatment groups were necropsied at the planned terminal sampling time points.

[0563] All the animals in vehicle and low dose Formula I (10 mg / kg) tolerated the dose well. No adverse effects were observed during the in-life period.

[0564] All the animals in the 30 mg / kg Formula I group were observed to have a face and genitalia swelling starting at 50 minutes after the initiation of IV infusion, which later resolved after the end of infusion. Animals in the dose groups of the toxicity portion (45 and 75 mg / kg) experienced swelling in face and prepuce swelling, tremor, inappetence and abnormal vocalisation during and after IV infusion. In addition, weak breathing, decreased body temperature, curled body position and loss of consciousness were observed.

[0565] Serum chemistry results in the 45 and 75 mg / kg groups showed increased levels of CRE, glucose and triglycerides compared to the control group. Significantly increased neutrophils and monocytes in these groups compared to the control suggested an immune response. Urine analysis revealed increased albumin at these high doses, suggesting kidney injury.

[0566] Dose-dependent test article-related increases in IL-6 and CXCL10 were noted in the study while no test article related changes to IL-10 or TNF-a were observed.

[0567] An increase in urinary nGAL at 30 m / kg was observed for up to 48 hours post-dose but it returned to normal levels by 72 hours post-dose. No other Formula I-related changes to urinary biomarkers were observed.

[0568] Gross necropsy in the 75 mg / kg group included observations of the spleen being mottled (red and dark red) and / or having a rough surface, and oedematous genitals. Furthermore, oedematous genital and red urine were each observed in 1 out of 3 animals at 45 mg / kg. Bone marrow smears indicated decreased myeloid cells at both 45 mg / kg and 75 mg / kg. In the toxicity portion of the study diffuse basophilic granules, ranging from mild to marked were observed in the cytoplasm of the tubular epithelial cells in the kidneys of Formula I-dosed animals. Additionally, minimal to mild multifocal degeneration / necrosis of the tubular epithelial cells were also observed. Due to the degenerative or necrotic change or high severity in the kidneys, the changes in these animals were considered to be adverse.

[0569] Example 16 - Repeat Dose GLP Toxicology of Formula I in Cynomolgus Monkeys The purpose of this GLP study was to evaluate the potential toxicity of Formula I following repeat dosing for 12 weeks and to assess the reversibility, persistence, or delayed occurrence of toxic effects following a 12-week recovery period in monkeys. Cynomolgus monkeys (n=4 / sex / group for main study and n=2 / sex / group in the control group and high dose group for recovery) were administered vehicle control, or Formula I at 4.8, 12 or 30 mg / kg / dose once every four weeks on Days 1, 29, 57 and 85 by IV infusion for 12 weeks (4 doses total).

[0570] Endpoint evaluations for both main study and recovery animals include clinical observations (including infusion site observation), body weight, food consumption, ophthalmology^ examination, organ weights, clinical pathology7(haematology7, serum chemistry, coagulation, urinalyses and urine chemistry), urinary biomarkers, cytokine analyses, and gross and microscopic pathology.

[0571] Blood samples were collected to allow for ADA analysis, if indicated, prior to dosing and on Day 85. Additionally, the TK of Formula I was evaluated and is described in Example 11. All animals survived to scheduled necropsy.

[0572] There were no test article-related changes observed at the infusion sites, in macroscopic observation, ophthalmologic examination, urine chemistry, levels of cytokines (IL-ip, TNF-a, IL-8, MCP-1, IFN-y), nor levels of urinary7CRE.

[0573] Formula I-related clinical observations included slight to moderate salivation and decreased activity7and multiple swellings in both sexes at 30 mg / kg / dose. These findings were noted on dosing day(s) and lasted for up to nine days after which all clinical observations were recovered. No test article-related clinical findings were observed in the recovery7phase.

[0574] Compared with concurrent controls, slightly lower body weight means were noted in both sexes at 30 mg / kg / dose on Days 35, 63 and / or 84. These differences were attributed to observed increases in body weight losses at this dose from Days 28 to 35, Days 56 to 63 and / or Days 77 to 84. At the end of the recovery7phase, the changes in mean body weight in males and body weight gain in both sexes were completely recovered. However, the mean body weight for females was not recovered.

[0575] Apparent Formula I-related transient inappetence was noted in all males and one female at 30 mg / kg / dose post the 3rd dose (males on Day 57. female on Days 57 to 58).

[0576] During the dosing phase, when compared with both concurrent controls and pretest data, apparent test article-related changes in haematology7included decreased erythrocyte count (RBC), hemoglobin (HGB) and HCT in males at 30 mg / kg / dose on Day 87 and increased absolute / percent monocytes (# / %MONO) in females at 30 mg / kg / dose on Day 3 and Day 87. All observed haematology changes were completely resolved by the end of the recovery phase.

[0577] When compared with both concurrent controls and pretest data, test article-related increases in FIB were observed in both sexes at 30 mg / kg / dose on both Days 3, and 87. Observed changes to FIB were completely resolved by the end of the recovery phase.

[0578] When compared with both concurrent controls and pretest data, test article-related increases in urea and CRE, and decreases in Mg were observed at 30 mg / kg / dose in females and / or males on Day 3 and Day 87, and a decrease in Cl was noted in females at 30 mg / kg / dose on Day 87. Urea was also increased in males at 12 mg / kg / dose on Day 87. In addition, apparent test article-related increases in AST were observed in females at 30 mg / kg / dose on Day 3 and in males at >12 mg / kg / dose on Day 87, and increases in ALT and CK were observed in females at 30 mg / kg / dose on Day 87.

[0579] Many of these changes listed above were associated with the degeneration / single cell necrosis and dilation / attenuation of the proximal convoluted tubules, basophilic granules, and hyaline cast formation indicated by microscopic examination. Besides the changes of Mg in females, all other changes were totally or partly recovered at the end of recovery phase.

[0580] On Day 3 and / or Day 87, apparent test article-related changes in urinalysis were observed at 30 mg / kg / dose including in positive leukocyte, positive protein, positive ketones and positive urobilinogen in males and / or females. These changes were associated with described changes in urea, CRE and Cl, as well as related to the renal injury indicated by microscopic examination. Positive occult blood was also observed in one male at 12 mg / kg / dose on Day 3 but was not observed in any other animals. This finding therefore lacked dose dependency. By the end of recovery phase, all urinalysis changes recovered.

[0581] Test article-related increases in IL-6 and CXCL10 (plasma cytokines), and Kim-1 and nGAL (urinary biomarkers) were observed in both sexes mostly at 30 mg / kg / dose. The increases in cytokine peaked at 2 hours post-dose with lower levels observed at 8 hours postdose. The increases in Kim-1 and nGAL peaked shortly after each Formula I administration and returned to baseline within 2 days and 4 weeks post-dose for Kim-1 and nGAL, respectively.

[0582] Formula Lrelated organ weight increases (mean absolute and / or relative) were observed in the liver of males and females, and the kidneys of females administered 30 mg / kg / dose. These changes were correlated with the microscopic findings of basophilic granules in the cytoplasm of Kupffer cells in the liver, and hyaline casts, proximal tubule single cell necrosis, tubular basophilic granules, mononuclear cell infiltration, and tubular dilation in the kidneys.

[0583] Test article-related histopathological findings were noted in the kidneys across all Formula I dose levels and in liver at 30 mg / kg / dose. Renal tubular basophilic granules of minimal (> 4.8 mg / kg / dose), mild (12 mg / kg / dose), and moderate (30 mg / kg / dose) severity were observed in both sexes. Mild hyaline cast formation, mononuclear cell infiltration and mild to moderate renal tubular dilation were noted in males and females in the high dose group. Moreover, minimal to mild proximal tubular degeneration / single cell necrosis was noted in males and females administered 12 mg / kg / dose. and moderate to marked proximal tubular degeneration / single cell necrosis was observed in both sexes at 30 mg / kg / dose.

[0584] Minimal and mild basophilic granules were noted in the cytoplasm of Kupffer cells of the liver in males and / or females given 30 mg / kg / dose. Histopathological findings in the liver and kidneys are consistent with the uptake and accumulation of oligonucleotides in endolysosomes and based on several research groups are not considered adverse.

[0585] At the end of the recovery' phase the only microscopic findings were minimal proximal tubular degeneration / single cell necrosis in one male and minimal renal tubular basophilic granules in two males administered 30 mg / kg / dose showing partial or full recovery' of all microscopic findings.

[0586] In conclusion. Formula I administered by IV infusion to Cynomolgus monkeys once every 4 yveeks for 12 yveeks at doses up to 30 mg / kg / dose yvas well tolerated with the low and mid dose groups (4.8 and 12 mg / kg / dose) not resulting in findings considered adverse. Under the conditions of the study, the NOAEL was considered to be 12 mg / kg / dose. The corresponding mean AUCo-49h and Cmax were 194000 h*ng / mL and 116000 ng / mL, respectively.

[0587] Example 17 - Clinical dose selection

[0588] As per FDA guidance for industry’ "‘Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” (FDA, 2005) for systemically administered drugs for deriving a human equivalent dose (HED) the No Observed Adverse Effects Levels (NOAELs) in the tested animal species should be normalised to body surface area. Therefore, to convert the doses determined for mouse and monkey in mg / kg to HED (assumes 60 kg human), the NOAELs for each species was divided by 12.3 and 3.1, respectively. The ongoing Phase 1 clinical study is comprised of a single ascending dose (SAD) component, yvith a MAD component planned to initiate after half of the SAD cohorts. The aim of the Phase 1 study is to evaluate the safety, tolerability, PD, and PK of single and subsequently multiple doses of IV administrations of Formula I. Nonclinical Support for Clinical Doses

[0589] With an estimated NOAEL at a dose level of 12 mg / kg in the GLP NHP (non-human primate) study, a corresponding human equivalent dose (HED) was 3.87 mg / kg. Additionally, the NOAEL in the mouse GLP study (data not shown) was determined to be 40 mg / kg resulting in a similar human equivalent total dose of 3.25 mg / kg.

[0590] Given that NHPs provide a more accurate prediction of human dose and safety outcomes due to their greater physiological and genetic resemblance to humans, NHPs were considered the more appropriate model for determining the maximum recommended starting dose (MRSD).

[0591] A retrospective analysis of renally cleared drugs showed that scaling using NHP best predicts human clearance of macromolecules drugs such as oligonucleotides.

[0592] Furthermore, the infusion regimen used in the NHP study instead of the bolus regimen used in the mouse study is the intended clinical regimen in the Phase 1 study.

[0593] Consistent with the FDA guidance, factoring a 10-fold safety window in the most appropriate species, z.e., NHP, the MRSD for clinical evaluation should be 0.4 mg / kg administered at 1-5 hour infusion. Initial clinical evaluation of the safety of Formula I was recommended at a starting dose of a single IV administration of 0.4 mg / kg.

[0594] Single Ascending Dose (SAD) (Part A) Study Rationale

[0595] In the ongoing Phase 1 study, Part A (SAD) Cohort 1, 2 and 3 investigating doses of 0.4 mg / kg, 1.2 mg / kg and 2.4 mg / kg Formula I, respectively in healthy volunteers, was completed. Part A (SAD) Cohort 4 investigating a dose of 4.0 mg / kg Formula I in healthy volunteers and Part B (SAD) Cohort 1 investigating a dose of 0.4 mg / kg in patients has commenced, with the remaining cohorts in Part B in patients, yet to initiate.

[0596] The PK parameters calculated for Part A (SAD) Cohorts 1-3 are presented in Table 4 below.

[0597] Table 4 Plasma Formula I Pharmacokinetic Parameters for Phase 1 Study (Part A)

[0598]

[0599] *n=6 Formula I and n=2 Placebo.

[0600] Abbreviations: AUCo-iast = AUC from time 0 to the time of the last quantifiable concentration; Cmax = Maximum observed concentration, occurring at Tmax; ND = Not determined; t> / 2= Half-life; Tmax = Time at which Cmax occurred.

[0601] Blood samples for the PK analysis of Part A (SAD) Cohorts 1, 2 and 3 were collected on Day 1 at multiple timepoints, and on Days 2. 3, 28, 56 and 84. At the NOAEL following a single dose of 40 mg / kg in the mouse on Day 1, the AUCo-48h was 58.800 h.ng / mL and Cmax was 129,000 ng / mL. At the NOAEL following a single dose of 12 mg / kg in the monkey on Day 1, the AUC0-673h was 111,000 h.ng / mL and Cmax was 57,400 ng / mL.

[0602] The margins between systemic exposure at the nonclinical NOAEL compared with the exposures obtained with the two completed Part A (SAD) Cohorts 1 and 2, are outlined in Table 5.

[0603]

[0604] Abbreviations: AUCo-iast = AUC from time 0 to the time of the last quantifiable concentration; Cmax = Maximum observed concentration, occurring at Tmax; ND = Not determined; t½ = Half-life; Tmax= Time at which Cmax occurred.

[0605] The mean plasma Formula I concentration-time profiles are shown in Figure 12.

[0606] Multiple Ascending Dose Study Rationale

[0607] In the ongoing Phase 1 study, Part C (MAD) will primarily investigate safety, tolerability, PK, and PD of Formula I up to 12 weeks via the IV route.

[0608] The dose selection for Part C (MAD) cohorts is supported by the results of the repeated dose toxicity studies in mice and monkeys whereby Formula I was administered by once monthly IV injection or infusion for a 12-week duration for a total of 4 doses. The NOAEL in the 12-week toxicology studies were considered to be 40 and 12 mg / kg / dose in mice and monkeys, respectively.

[0609] It was previously established that the monkey was the most appropriate species, and therefore considered to support the human dose selection. At the NOAEL of 12 mg / kg in the monkey, an AUCo-49h and Cmax of 194000 h*ng / mL and 116000 ng / mL was determined.

[0610] Based on the systemic exposure values obtained in Part A (SAD) of the ongoing Phase 1 study at 0.4 mg Formula I (which is the proposed starting dose for Part C (MAD) Cohort 1 -with AUC0-last of 2315 ng*h / mL and Cmax of 2688 ng / mL), there is an exposure margin of 83.8 fold based on AUC0-last and a 43.2 fold margin based on Cmax comparisons to the monkey values obtained at the NOAEL on Day 85.

[0611] Additionally, utilising the EIED calculation with the most appropriate species (monkey) a NOAEL of 12 mg / kg is equivalent to 3.87 mg / kg. With a 10-fold safety margin, the MRSD is 0.387 which is supportive of a MAD starting dose of 0.4 mg / kg by IV administration.

[0612] Collectively, the nonclinical studies performed to date together with the currently available clinical data from Part A (SAD) Cohorts 1, 2 and 3, support the initiation of Part C (MAD) of the ongoing Phase 1 study.

[0613] Example 18 - Formula I formulation pH stability testing and buffer optimization

[0614] Stability of Formula I in Water

[0615] The goal of this study was to determine whether water may act as a suitable vehicle for Formula I formulations. 1 mg / mL solutions of Formula I in water were prepared and incubated at various temperatures for seven days. It is evident from Table 6 that water alone was not a suitable vehicle for Formula I.

[0616]

[0617] Stability of Formula I in Buffered Solution of Different pH

[0618] The goal of this study was to determine the optimal pH value for buffered solutions of Formula I. Solutions of Formula I (1 mg / mL) in 10 mM sodium phosphate buffer, 0.85% NaCl were prepared and the pH adjusted to values between pH 6.0 and pH 8.0. A buffer of 0.9% NaCl (saline) was also prepared. Solutions were incubated under accelerated conditions (50 °C) and appearance, purity and assay evaluated for up to six days. At all timepoints and under all conditions, solutions were clear, colourless solutions. From the data shown in Table 7, it w as shown that 10 mM phosphate buffer at pH 6.0 is the optimal buffer for Formula I. Table 7 - Stability of Formula I in Buffered Solution of Different pH

[0619]

[0620]

[0621] Formula I Drug Substance - Stability

[0622] The compound was shown to be stable under long-term and accelerated storage conditions. Key stability data are shown in Tables 8 and 9 below:

[0623]

[0624]

[0625] Formula I Drug Product - Buffer Composition

[0626] Formula I is supplied at a strength of 20 mg / mL, packaged in Type I size 10R glass vials, with chlorobutyl rubber stoppers. The composition of this formulation and its stability are shown in Tables 10 and 11, respectively.

[0627]

[0628]

[0629] APPENDIX

[0630] Table 12: Sequences

[0631]

Claims

CLAIMS1. A method for treating autosomal dominant polycystic kidney disease (ADPKD) in a subject, the method comprising systemically administering to the subject a conjugate comprising an antisense oligonucleotide (ASO) that binds to a targeted portion of the 3' untranslated region (UTR) of a PKD1 mRNA and reduces specific binding of a miR- 17 family member to the 3' UTR and a cell penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 13.

2. A method for increasing expression of PKD1 transcript or PCI protein in kidney cells of a subject, the method comprising systemically administering to a subject suffering from ADPKD a conjugate comprising an antisense oligonucleotide (ASO) that binds to a targeted portion of the 3' untranslated region (UTR) oiaPKDl mRNA and reduces specific binding of a miR-17 family member to the 3' UTR or comprises the sequence set forth in any one of SEQ ID NOs: 1-12 and a cell penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 13.

3. The method of claim 1 or claim 2, wherein the expression of the PKD1 transcript or PCI protein is increased in the kidney at least 1.1 fold.

4. The method of any one of claims 1 to 3, wherein the ASO comprises the sequence set forth in SEQ ID NO: 1.

5. The method of any one of claims 1 to 4 comprising administering 0.4-5.0 mg / kg of the conjugate.

6. The method of any one of claims 1 to 5, comprising administering the conjugate intravenously.

7. The method of any one of claims 1 to 6, wherein following administration a therapeutically effective amount of the conjugate is detectable in a kidney and, optionally, liver of the subject.

8. The method of claim 7, wherein following administration, a therapeutically effective amount of the conjugate is detectable in a cyst in a kidney of the subject.

9. The method of claim 7 or claim 8. wherein the therapeutically effective amount is detected in a kidney and, optionally in a liver in a model organism administered a dose of theconjugate that accounts for the difference in the model organism to the subject to demonstrate detection of the conjugate in the kidney and, optionally in a liver in the subject.

10. The method of any one of claims 5 to 9, comprising administering 0.4-4.0 mg / kg of the conjugate.

11. The method of claim 10, comprising administering 1.2-2.4 mg / kg of the conjugate.

12. The method of any one of claims 1 to 11, comprising administering multiple doses of the conjugate.

13. The method of claim 12, comprising administering the conjugate to the subject at least every four to twelve weeks.

14. The method of claim 12 comprising administering the conjugate to the subject at least every' four weeks.

15. The method of any one of claims 1 to 14, wherein the CPP in the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 13.

16. The method of claim 16, wherein each amino acid in the CPP is a D amino acid.

17. The method of any one of claims 1 to 16, wherein the nucleotide sequence of the ASO in the conjugate consists of the sequence set forth in SEQ ID NO: 1.

18. The method of according to any one of claims 1 to 17 wherein the antisense oligonucleotide comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage or wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino linkage.

19. The method of claim 18, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide.

20. A method for delivering a compound to a kidney or liver of a subject, the method comprising systemically administering a conjugate comprising the compound conjugated to a cell penetrating peptide (CPP) comprising the sequence set forth in SEQ ID NO: 13.

21. The method of claim 20, comprising administering the conjugate intravenously.

22. The method according to any one of claims I to 19. wherein target engagement of the conjugate in kidney cells of the subject is at least about 38%.

23. The method according to claim 22, wherein the target engagement is at least about 50%.

24. A conjugate comprising an antisense oligonucleotide (ASO) the nucleotide sequence of which consists of the sequence set forth in SEQ ID NO:1 and a cell penetrating peptide (CPP) the amino acid sequence of which consists of the sequence set forth in SEQ ID NO: 13.

25. A pharmaceutical composition comprising the conjugate according to claim 24 and a pharmaceutically acceptable excipient.

26. A container comprising the conjugate of claim 24 or the pharmaceutical composition of claim 25.

27. A pharmaceutical composition comprising:(i) a conjugate comprising a cell penetrating peptide (CPP) the amino acid sequence of which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 in which each amino acid is a D amino acid and a phosphorodiamidate morpholino oligonucleotide the nucleotide sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 1; and(ii) a physiological buffer effective to maintain the pharmaceutical composition at about pH 6.0.

28. The pharmaceutical composition according to claim 27, wherein the physiological buffer is a phosphate buffer.

29. The pharmaceutical composition according to claim 27 or claim 28, having osmolality from 260 to 350 mOsm / kg.

30. The pharmaceutical composition according to any one of claims 27 to 29, further comprising a tonicity modifier.

31. The pharmaceutical composition according to claim 30. wherein the tonicity modifier is sodium chloride.

32. The pharmaceutical composition according to any one of claims 27 to 31, wherein the amino acid sequence of the cell penetrating peptide consists of the amino acid sequence set forth in SEQ ID NO: 13 in which each amino acid is a D amino acid and the nucleotide sequence of the phosphorodiamidate morpholino oligonucleotide consists of the nucleotide sequence set forth in SEQ ID NO: 1.33 The pharmaceutical composition according to any one of claims 27 to 31, wherein the concentration of the conjugate is 10 mg / ml to 30 mg / mL.

34. The pharmaceutical composition according to claim 33, wherein the concentration of the conjugate is about 20 mg / ml.

35. A lyophilized composition comprising a cell penetrating peptide (CPP) the amino acid sequence of which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 in which each amino acid is a D amino acid and a phosphorodiamidate morpholino oligonucleotide the nucleotide sequence of which comprises or consists of the sequence set forth in SEQ ID NO: 1.

36. The lyophilized composition according to claim 35. wherein the amino acid sequence of the CPP consists of the amino acid sequence set forth in SEQ NO: 13 in which each amino acid is a D amino acid and the nucleotide sequence of the phosphorodiamidate morpholino oligonucleotide consists of the nucleotide sequence set forth in SEQ ID NO: 1.

37. The method according to any one of claims 1 to 23; the conjugate according to claim 24; the pharmaceutical composition according to any one of claims 25 to 34; or the lyophilized composition according to claim 35 or claim 36, wherein the conjugate corresponds to formula Formula I set forth in figure 11.

38. The method according to any one of claims 1 to 23; the conjugate according to claim 24; the pharmaceutical composition according to any one of claims 25 to 34; or the lyophilized composition according to claim 35 or claim 36, wherein the conjugate comprises a flexible linker linking the CPP to the phosphorodiamidate morpholino oligonucleotide, preferably wherein the amino acid sequence of the flexible linker only includes D-amino acids.

39. The method, the conjugate, the pharmaceutical composition, or the lyophilized composition according to claim 38, wherein the C-terminus of the CPP is linked to the 3' end of the phosphorodiamidate morpholino oligonucleotide via the flexible linker.

40. The method, the conjugate, the pharmaceutical composition, or the lyophilized composition according to claim 38 or claim 39, wherein the ammo acid sequence of the flexible linker comprises or consists of an amino acid sequence selected from the group consisting of: GGGGS. GGGGSGGGGS (SEQ ID NO: 14), GAS, GGG, GSG. GTG, GGTAGSTGG (SEQ ID NO: 15), GASGGASG (SEQ ID NO: 16) and GASG.

41. The method, the conjugate, the pharmaceutical composition, or the lyophilized composition according to claim 40, wherein the amino acid sequence of the flexible linker consists of the amino acid sequence set forth in SEQ ID NO: 16.

42. A vial comprising the pharmaceutical composition according to any one of claims 25 to 34 or 37 to 41; or the lyophilized composition according to any one of claims 35 to 41.

43. The vial according to claim 42, wherein the vial is a glass vial.

44. The vial according to claim 42 or claim 43, comprising an elastomeric stopper.

45. The vial according to any one of claims 42 to 44, wherein the vial comprises a fill volume of 5 mL to 15 mL.

46. The vial according to claim 45. wherein the vial comprises a fill volume of 10 mL.

47. The vial according to any one of claims 42 to 46, comprising the pharmaceutical composition in a volume in the range of 5 mL to 15 mL.

48. The vial according to claim 48, comprising the pharmaceutical composition in a volume of 10 mL.

49. The vial according to any one of claims 42 to 48 comprising the conjugate at a concentration of 10 to 30 mg / mL.

50. The vial according to any one of claims 42 to 48 comprising the conjugate at a concentration of 20 mg / mL. YYY

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