Methods for treating, alleviating, and / or preventing polycystic kidney and polycystic liver

Abstract

Described herein is a method of alleviating and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound that suppresses translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene. TIFF2025519109000063.tif105133

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 345,634, filed May 25, 2022, and U.S. Provisional Patent Application No. 63 / 359,109, filed Jul. 7, 2022, each of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under grant 1K08DK119642 - 01 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing An ASCII text file, created on May 24, 2023, and named "047162 - 7395WO1(02006)_Seq Listing.xml", consisting of 173 kilobytes, is hereby incorporated by reference in its entirety.

Background Art

[0004] Background Autosomal dominant polycystic kidney disease (ADPKD) is a highly penetrant hereditary polycystic disease that typically causes cysts and deformities in the kidneys over a span of decades, ultimately leading to kidney failure that requires dialysis or transplantation in the majority of patients after the age of 40.

[0005] ADPKD is sometimes considered a rare disease, but the number of ADPKD patients is actually quite large. It is estimated that there are over 600,000 ADPKD patients in the United States alone and over 12 million worldwide. Moreover, since ADPKD is not the subject of founder mutations but rather de novo mutations that occur constantly, the ADPKD patient population is expected to further increase as the world population grows.

[0006] Currently, the only approved drug for ADPKD is tolvaptan. Unfortunately, tolvaptan has serious side effects. The drug causes polyuria (6.0 ± 1.8 L of urine per day, Kramers et al., BMC Nephrol. 2018; 19: 157 (Non-Patent Document 1)), has a black box warning regarding liver injury (i.e., the most severe warning by the FDA for a marketed drug), and is subject to a risk evaluation and mitigation strategy (REMS) by the FDA. Considering that many ADPKD patients require long-term treatment, the serious side effects of tolvaptan are particularly undesirable. Furthermore, even when tolerated at the maximum therapeutic dose, tolvaptan therapy only provides a very slight delay in renal failure.

[0007] More than 90% of patients with ADPKD also have numerous cysts in the liver, which is known as polycystic liver disease. These cysts occur due to a mechanism similar to that which affects renal tubular epithelium, affecting the bile duct epithelium. Hepatic cysts typically do not cause liver failure, but a subset of patients may have debilitating symptoms, including pain, infections, swelling, and early satiety that prevents adequate nutrition, due to significant enlargement of the liver. There is no FDA-approved drug therapy for hepatic cysts. Instead, patients are subject to repeated interventions or surgical procedures to aspirate, fenestrate, or excise the cysts, or to undergo partial or total hepatectomy with liver transplantation. Clinically indistinguishable polycystic liver disease can occur in the absence of renal cysts when caused by different but related genetic mechanisms. This isolated polycystic liver disease (also known as PCLD, "autosomal dominant polycystic liver disease" or "ADPLD") is considered a rare genetic disorder, but its prevalence may approach that of ADPKD when asymptomatic cases detected at autopsy are included. Approximately 20% of PCLD patients develop overt clinical symptoms such as dyspnea, early satiety, abdominal distension, malnutrition, gastroesophageal reflux disease, and back pain, caused by compression of adjacent organs due to liver enlargement or cyst complications. Currently, the only definitive treatment for PCLD, which is used only in the most severe cases, is liver transplantation.

[0008] Therefore, new treatment methods for polycystic kidney and polycystic liver are needed. The present invention addresses this need.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

[0010] Summary In some aspects, the present invention is directed to the following.

[0011] In one aspect, the present invention provides a method of treating, alleviating, and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that suppresses translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene.

[0012] In various embodiments, the method is a method of treating, alleviating, and / or preventing ADPKD in a subject, wherein the ADPKD is caused by or associated with a mutation in the PKD1 gene in the subject.

[0013] In various embodiments, the method is a method of treating, alleviating, and / or preventing PCLD in a subject, wherein the PCLD is caused by or associated with a germline mutation in the PKD1 gene, the PKD2 gene, the PRKCSH gene, the SEC63 gene, the GANAB gene, the ALG8 gene, the ALG9 gene, the SEC61B gene, or the DNAJB11 gene in the subject.

[0014] In various embodiments, the compound comprises: CRISPR components that disrupt the genomic DNA sequence encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the CRISPR components; or Antisense oligonucleotides (ASOs) that block translation of the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the ASOs.

[0015] In various embodiments, the compound comprises a CRISPR component or an expression vector that expresses a CRISPR component, wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF.

[0016] In various embodiments, the compound comprises an ASO or an expression vector that expresses an ASO, wherein the portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, e.g., within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0017] In various embodiments, the length of the ASO is 10 nucleotides or more, such as 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

[0018] In various embodiments, the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0019] In various embodiments, at least one of the following applies: (a) The ASO is perfectly complementary to one of the sequences set forth in SEQ ID NOs: 14 - 61, (b) The ASO is perfectly complementary to one of the sequences set forth in SEQ ID NOs: 62 - 109, (c) The ASO is perfectly complementary to one of the sequences set forth in SEQ ID NOs: 110 - 157, (d) The ASO has a nucleotide sequence TIFF2025519109000002.tif4128, nucleotide sequence including TIFF2025519109000003.tif4128.

[0020] In various embodiments, the ASO includes a modified nucleobase, a modified sugar moiety, or a modified linkage.

[0021] In various embodiments, at least one of the following applies: (a) The ASO includes a modified sugar moiety, and the modified sugar moiety includes a 2'-O-methylated modified ribose group such as a 2'-O-methylated ribose group. (b) The ASO includes a modified linkage, and the modified linkage includes a phosphorothioate (PS) linkage.

[0022] In various embodiments, the subject is a mammal such as a human.

[0023] In various embodiments, the compound includes an ASO or an expression vector expressing the ASO, wherein the concentration of the ASO in the kidney or lung of the subject is in the range of about 1 nm to about 100 nm.

[0024] In another aspect, the present invention provides a method for increasing PKD1 expression in a cell, comprising contacting the cell with an effective amount of a compound that suppresses the translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene.

[0025] In various embodiments, the cell has a mutation in the PKD1 gene, PKD2 gene, PRKCSH gene, SEC63 gene, GANAB gene, ALG8 gene, ALG9 gene, SEC61B gene, or DNAJB11 gene.

[0026] In various embodiments, the cell is in a tissue or a subject.

[0027] In various aspects, the cell is a renal cell in a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or a hepatocyte in a subject diagnosed with polycystic liver disease (PCLD).

[0028] In various aspects, the compound comprises: A CRISPR component that disrupts the genomic DNA sequence encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the CRISPR component; or An antisense oligonucleotide (ASO) that blocks the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the ASO.

[0029] In various aspects, the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF.

[0030] In various aspects, the compound comprises an ASO or an expression vector expressing the ASO, wherein the portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, e.g., within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0031] In various aspects, the length of the ASO is 10 nucleotides or more, such as 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

[0032] In various embodiments, the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0033] In various embodiments, at least one of the following applies: (a) The ASO is fully complementary to one of the sequences set forth in SEQ ID NOs: 14-61, (b) The ASO is fully complementary to one of the sequences set forth in SEQ ID NOs: 62-109, (c) The ASO is fully complementary to one of the sequences set forth in SEQ ID NOs: 110-157, (d) The ASO contains the nucleotide sequence TIFF2025519109000004.tif4128, the nucleotide sequence TIFF2025519109000005.tif4128.

[0034] In various embodiments, the ASO contains a modified nucleobase, a modified sugar group, or a modified linkage.

[0035] In various embodiments, at least one of the following applies: (a) The ASO contains a modified sugar, and the modified sugar group contains a 2'-O-methylated modified sugar group such as a 2'-O-methylated ribose group, (b) The ASO contains a modified linkage, and the modified linkage contains a phosphorothioate (PS) linkage.

[0036] In various embodiments, the compound contains an ASO or an expression vector that expresses the ASO, wherein the concentration of the ASO contacted with the cell ranges from about 1 nm to about 100 nm.

Brief Description of the Drawings

[0037] The following detailed description of illustrative embodiments will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration, non-limiting embodiments are shown in the drawings. However, it should be understood that the specification is not limited to the exact arrangements and instrumentalities shown in the drawings.

[0038] (FIGS. 1A-1C) Demonstrate, according to some embodiments, that renal cysts are caused by a reduced functional dosage of PKD1 / polycystin-1 (PC1) due to either a loss-of-function mutation in the PKD1 gene itself or a mutation in the endoplasmic reticulum (ER) gene responsible for PC1 maturation, and that this can be rescued by increasing the Pkd1 copy number. FIGS. 1A-1B: MRI of severe polycystic disease in patients with PKD1 non-truncating mutations (kidney and liver) (FIG. 1A) or SEC63 mutations (liver only) (FIG. 1B). FIG. 1C: 3-week-old PC1 gene dosage-dependent mouse model. PC1 matures inefficiently in the ER in the absence of Sec63, but in this situation, additional cyst formation can be prevented by producing more PC1 from three additional genomic copies of Pkd1 (Pkd1 F / H -BAC). Ksp-Cre is active in the distal nephron from mid-embryonic development. (FIGS. 2A-2B) Illustrate certain aspects of the 5'UTR and uORF of human PKD1, according to some embodiments. FIG. 2A: Linear sequence of the start of the PKD1 mRNA sequence showing the position of the uORF in the 5'UTR. FIG. 2B: Predicted secondary structure of the PKD1 5'UTR. FIG. 2A shows nucleotides 1-300 of SEQ ID NO:1, which includes the coding sequence for the first 30 amino acid residues of the human PC-1 protein TIFF2025519109000006.tif4128 is included. FIG. 2B shows nucleotides 1-209 of SEQ ID NO:1. (FIGS. 3A-3B) Illustrate certain aspects regarding how uORFs function and how antisense oligonucleotides (ASOs) inhibit uORFs according to some embodiments. FIG. 3A: Illustrates the detrimental effect of uORFs on protein translation. FIG. 3B: ASO that prevents uORF translation and allows for increased protein translation. (FIGS. 4A-4D) Demonstrate according to some embodiments that uORFs in the 5'UTR of human PKD1 downregulate translation and that this downregulation is abolished by disruption of the uORFs. FIG. 4A: Dual luciferase reporter construct in which the 5'UTR of Renilla luciferase is replaced by either the wild-type 5'UTR or a variant of the human PKD1 5'UTR with a mutant uORF. FIG. 4B: Sequences of the wild-type uORF (“wt”), and mutant uORFs containing either one single-base edit (“ΔuORF1” and “ΔuORF2”) or two single-base edits (“ΔuORF1&2”) to abolish either one or both of the uORFs. In FIG. 4B, ΔuORF1 means the mutation from A to T at nucleotide residue 123 of SEQ ID NO:2, ΔuORF2 means the mutation from A to T at nucleotide residue 190 of SEQ ID NO:2, and ΔuORF1&2 means that both A to T mutations are included. FIG. 4C: Targeted editing of the PKD1 5'UTR resulted in a significantly increased expression of the luciferase protein. Notably, abolishing both uORFs increased protein expression nearly 4-fold (“1&2” vs. “wt”). FIG. 4D: Mutations in the uORFs did not change mRNA expression. (Figure 5) Demonstrate that an antisense oligonucleotide (ASO) that inhibits the uORF in the 5'UTR of PKD1 increases PC1 protein expression. Cells expressing the wt-uORF luciferase reporter (Figures 4A-4D) were transfected and then incubated with nucleotide ASO that is complementary to the mRNA sequence upstream of the ATG of the uORF1, uORF2, or both uORF start codons and overlaps with the ATG of the start codon, or a mismatch control (MM1, MM2) ASO, together with the transfected cells. Lysates collected after 24 hours of treatment showed a significant increase (approximately 3-fold increase) in luciferase expression when both uORF1 and uORF2 were targeted. (Figures 6A-6G) Demonstrate that ASO treatment in the culture medium of human epithelial cells significantly increased the protein expression of PC1 according to some embodiments. Figure 6A: ASO1, ASO2, and their combinations increase the steady-state expression of PC1 without effecting mRNA expression. The protein expression data in this figure are from the 48-hour time point also included in Figure 6D. Figures 6B-6D: The increase in PC1 protein expression achieved by treatment with ASO1, ASO2, or their combination is most prominent when the treatment is maintained longer (the expression levels normalized to vinculin, a loading control in Western blot, indicate that the 48-hour and 96-hour time points have the most potent effects). It should be noted that fresh medium containing a defined concentration of ASO was applied once a day. Figures 6E-6G Various dosages of ASO1 and ASO2 produced an expression-increasing effect in cultured epithelial cells. (Figure 7) Demonstrate that mouse polycystic kidney disease caused by epithelial cell-specific loss of the human polycystic kidney gene DNAJB11 is sensitive to a 50% reduction in PC1 gene dosage according to some embodiments. This genetic interaction supports the hypothesis that a 2-fold increase in PC1 expression achieved in in vitro preliminary data may not only slow down but also be sufficient to completely prevent cyst formation. (Figure 8) According to some embodiments, uORF1 and uORF2 in the mouse Pkd1 mRNA 5'UTR are shown. The mouse PKD1 5'UTR contains two potential uORFs starting with ATG that correspond to those found in the human sequence. These two uORFs are aligned with the above human uORFs and named mouse uORF1 and mouse uORF2. Figure 8 shows residues 1 to 360 of SEQ ID NO:7, which includes the coding sequence for the first 16 amino acid residues of the mouse PC1 protein TIFF2025519109000007.tif4128 is included. (Figure 9) According to some embodiments, the design of an ASO for sterically blocking the translation of the mouse Pkd1 uORF is shown. Figure 9 shows nucleotide residues 1 to 316 of SEQ ID NO:7. (Figures 10A - 10B) PC1 protein expression upon treatment with an ASO targeting the mouse Pkd1 uORF in a mouse cell line containing an HA epitope tag on the C-terminus of Pkd1 to enable assessment of the C-terminal fragment, according to some embodiments. A concentration of 20 nM was used for all ASOs. Assessment at (Figure 10A) 24 hours or (Figure 10B) 96 hours after administration. (Figure 11) PC1 protein expression upon treatment with an ASO targeting the mouse Pkd1 uORF in a mouse cell line containing a missense mutation p.R2216W (ortholog of the human variant p.R2220W) and a V5 epitope tag on the C-terminus of Pkd1 to enable assessment of the C-terminal fragment, according to some embodiments. A concentration of 20 nM was used for all ASOs; assessment 96 hours after administration. The data demonstrate that inhibition of the Pkd1 uORF significantly increases the production of the mature protein (PC1 C-terminal fragment), even in the context of a pathogenic missense variant. (Figure 12) shows the positions of three translation start sites seen in publicly available ribosome profiling experiments visualized by publicly available Ribo - uORF resources. The uORF with the most evidence of translation in the most experiments was our uORF1. Two additional uORFs (uORF3 and uORF4) with CTG start codons in the human PKD1 mRNA 5'-UTR were also identified by evidence from at least one ribosome profiling data set. According to some embodiments, these can be targeted by ASOs to increase PC1 protein expression. The sequence shown in Figure 12 is nucleotides 1 - 306 of SEQ ID NO:1 (having DNA nucleotide T instead of RNA nucleotide U). The uORF numbering has been edited from the website default to match ours (see Liu et al. Nucleic Acids Research 2023). **Modes for Carrying Out the Invention**

[0039] **Detailed Description** The following disclosure provides many different embodiments or examples for realizing different characteristics of the provided subject matter. To simplify the disclosure of the present invention, specific examples of components and arrangements are described below. Of course, these are merely examples and are not intended to be limiting. For example, forming a first characteristic above or on a second characteristic in the following description may include embodiments where the first and second characteristics are formed in direct contact, and may also include embodiments where additional characteristics are formed between the first and second characteristics so that the first and second characteristics cannot be in direct contact. Additionally, in the disclosure of the present invention, reference numerals and / or letters may be repeated in various examples. This repetition is for the purpose of simplicity and clarity and does not by itself determine the relationship between the various embodiments and / or configurations being discussed.

[0040] In addition to PCLD, a significant subset of ADPKD is caused by insufficient functional gene dosage of PC1, and these are caused by either loss-of-function mutations in the PKD1 gene itself or mutations in endoplasmic reticulum (ER) genes responsible for sorting and transporting PKD1 to the cell surface of primary cilia.

[0041] The hypothesis has been proposed that both PCLD and ADPKD caused by insufficient functional gene dosage of PC1 can be treated, alleviated, and / or prevented by increasing the functional gene dosage of PC1 in patients. The only known method for increasing PC1 expression is the inhibition of microRNA 17 (Lakhia et al., “PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression”). The basis of the proposed therapy, which is also at the preclinical investigation level in a mouse model, is to block the binding of microRNA 17 to the 3'-UTR of the PKD1 transcript, which the authors propose inhibits the steady state of PKD1 mRNA. In vivo studies have shown that the approach of therapeutically increasing the PC1 gene dosage is promising. Since complementary sequences to the microRNA 17 sequence are found in many genes, treating PCLD and ADPKD with this molecule is expected to impose a significant and unpredictable burden of off-target effects, i.e., loss of specificity.

[0042] In this study, four upstream open reading frames (uORFs) were discovered in the 5'-untranslated region (5'-UTR) of PKD1 messenger RNA (mRNA). In this study, it was further found that translation of uORF1 and / or uORF2 results in a reduction in the translation of PKD1 mRNA into the PC1 protein, and that translation of PKD1 mRNA is significantly enhanced either by mutating these two uORFs to deletion or by using antisense oligonucleotides (ASOs) to block the translation of the uORFs, thereby increasing the protein level of PC1. It is expected that the third and fourth uORFs may function similarly to or complementarily to the first and second uORFs, and that similar or enhanced results can be achieved by deletion and / or suppression of the third and fourth uORFs. Since the uORFs in the 5'-UTR of PKD1 have unique sequences and methods targeting uORF sequences such as CRISPR or ASO can be designed with high specificity, treatment of ADPKD or PCLD through uORFs is expected to be highly specific.

[0043] Accordingly, in some aspects, the invention is directed to methods of treating, alleviating, and / or preventing polycystic kidney or polycystic liver.

[0044] In some aspects, the invention is directed to methods for increasing the functional gene dosage of PC1 in a cell, tissue, or subject.

[0045] Definitions As used herein, each of the following terms has the meaning associated with it in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the nomenclature used herein, as well as the experimental procedures in veterinary pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry, are well known and commonly employed in the art. It should be understood that the order of steps or the order for performing a particular operation is not important as long as the teachings of the present invention remain operable. The use of any section headings is intended to assist in reading the document and should not be construed as limiting; information relevant to a section heading may appear within or outside that particular section. All publications, patents, and patent documents referred to in this document are hereby incorporated by reference in their entirety as if each were individually incorporated by reference.

[0046] In this application, when an element or component is said to be included in and / or selected from a recited list of elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0047] In the methods described herein, acts can be performed in any order, except when a temporal or operational order is explicitly recited. Moreover, the specified acts can be performed simultaneously unless the explicit language of the claims states that they are to be performed separately. For example, the claimed act of doing X and the claimed act of doing Y can be carried out simultaneously in a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0048] As used herein, unless the context clearly dictates otherwise, the terms "a," "an," or "the" are used to include one or more than one. The term "or" is used to mean a non-exclusive "or" unless otherwise indicated. The statements "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B."

[0049] As used herein when referring to a measurable value such as an amount, duration of time, etc., "about" means within ±20%, or ±10% in certain aspects, ±5% in certain aspects, ±1% in certain aspects, or ±0.1% in certain aspects of the specified value, because such variations are appropriate to practice the methods of this disclosure.

[0050] As used herein, "PKD1" refers to the human gene transcribed to produce the mRNA product represented by SEQ ID NO:1, all human genes that are allelic to the human gene that produces the mRNA of SEQ ID NO:1; all ortholog genes in non-human species, as well as all mRNA and protein products of human and non-human genes (which are sometimes referred to in both the art and this specification as "polycystin-1," "polycystin 1," "PC-1," or "PC1" proteins). TIFF2025519109000008.tif164161TIFF2025519109000009.tif221161TIFF2025519109000010.tif221161TIFF2025519109000011.tif221161TIFF2025519109000012.tif221161TIFF2025519109000013.tif221161TIFF2025519109000014.tif221161TIFF2025519109000015.tif221161TIFF2025519109000016.tif221161TIFF2025519109000017.tif79161

[0051] In some embodiments, PKD1 refers to human PKD1 having a 5'UTR with the RNA sequence set forth in SEQ ID NO:2. TIFF2025519109000018.tif44161

[0052] In some embodiments, PKD1 refers to human PKD1 having the following upstream open reading frame in its 5'UTR. TIFF2025519109000019.tif102161

[0053] In some embodiments, Pkd1 refers to mouse Pkd1 having the following mRNA sequence. TIFF2025519109000020.tif150161TIFF2025519109000021.tif221161TIFF2025519109000022.tif221161TIFF2025519109000023.tif221161TIFF2025519109000024.tif221161TIFF2025519109000025.tif221161TIFF2025519109000026.tif221161TIFF2025519109000027.tif221161TIFF2025519109000028.tif221161TIFF2025519109000029.tif107161

[0054] In some embodiments, Pkd1 refers to mouse Pkd1 having a 5'UTR with the RNA sequence set forth in SEQ ID NO:8. TIFF2025519109000030.tif58161

[0055] In some embodiments, Pkd1 refers to mouse Pkd1 having the following upstream open reading frame in its 5'UTR. TIFF2025519109000031.tif30161

[0056] For the purposes of this specification, the terms "first uORF", "second uORF", "third uORF", or "fourth uORF" do not mean that these uORFs are located in the mRNA sequence in that order. Rather, the terms "first uORF", "second uORF", "third uORF", and "fourth uORF" refer to uORFs in the 5'UTR of PKD1 mRNA having the above sequence, or uORFs at the same or similar positions in the 5'UTR of PKD1 mRNA and having a homologous sequence (such as having at least about 80%, 85%, 90%, or 95% sequence identity).

[0057] Abbreviations: 5'UTR: 5'-untranslated region. ADPKD: autosomal dominant polycystic kidney disease. ADPLD: autosomal dominant polycystic liver disease. ASO: antisense oligonucleotide. ER: endoplasmic reticulum. PCLD: polycystic liver disease. PKD: polycystic kidney disease. uORF: upstream open reading frame.

[0058] Methods for treating, alleviating, and / or preventing ADPKD or PCLD In some aspects, the present invention is directed to methods for treating, alleviating, and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof.

[0059] In some embodiments, the method comprises administering to a subject an effective amount of a compound that suppresses the translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, or the fourth uORF of the PKD1 gene, or combinations thereof, or affects these secondary mRNA structures.

[0060] In some embodiments, the method is a method of treating, alleviating, and / or preventing ADPKD in a subject, where ADPKD is caused by or associated with a mutation in the PKD1 or PKD2 gene in the subject.

[0061] In some embodiments, the method is a method of treating, alleviating, and / or preventing PCLD in a subject, where PCLD is caused by or associated with a germline mutation in the PKD1 gene, PKD2 gene, PRKCSH gene, SEC63 gene, GANAB gene, ALG8 gene, ALG9 gene, SEC61B gene, or DNAJB11 gene, or other human disease genes not yet established for the ADPKD-PCLD disease spectrum in the subject.

[0062] In some embodiments, compounds that suppress the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene include: CRISPR components that disrupt the genomic DNA sequence encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the CRISPR components; or Antisense oligonucleotides (ASOs) that block the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the ASOs.

[0063] In this study, the sequences of four uORFs in the 5'-UTR of PKD1 mRNA were identified, and it was confirmed that the expression of PC1 could be significantly increased by suppressing either or both of the first two uORFs by either modifying the sequences of the uORFs or blocking the uORFs with ASOs. It is expected that the third and fourth uORFs in PKD1 mRNA may function similarly to or complementarily to the first and second uORFs, and that similar or enhanced results can be achieved by deleting or suppressing uORF3 and 4.

[0064] Those skilled in the art will understand that the techniques for designing both the CRISPR system and ASOs are known in the art, and that using the target sequences of the uORFs identified by this study and the descriptions of CRISPR and ASOs in this specification (e.g., the sections "Suppression of PKD1 uORF by CRISPR Technique" and "Suppression of PKD1 uORF by ASO Technique"), CRISPR components and ASOs can be designed and obtained without undue experimentation.

[0065] In some embodiments, the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF.

[0066] In some embodiments, the compound comprises an ASO or an expression vector that expresses an ASO, and the portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, for example within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0067] In some embodiments, the ASO suppresses the first uORF (“uORF 1”). In some embodiments, the ASO targets the entire or a continuous portion of TIFF2025519109000032.tif3128 (SEQ ID NO:13, with the start codon highlighted), which includes the 5' portion of uORF 1 and several nucleotides up to its 5' end.

[0068] In some embodiments, the sequence of the 5'-UTR that is complementary to the ASO (such as about 80% or more complementary, about 85% or more complementary, about 90% or more complementary, about 95% or more complementary, or completely complementary) and targeted by the ASO is at least one of the following. TIFF2025519109000033.tif102161TIFF2025519109000034.tif219161TIFF2025519109000035.tif52161

[0069] In some embodiments, the ASO suppresses uORF 2. In some embodiments, the ASO targets the entire or a portion of the sequence TIFF2025519109000036.tif3128 (SEQ ID NO:, with the start codon highlighted), which includes the 5' portion of uORF 2 and several nucleotides up to its 5' end.

[0070] In some embodiments, the sequence of the 5'-UTR that is complementary to the ASO (complementary by about 80% or more, about 85% or more, about 90% or more, about 95% or more, or completely complementary, etc.) and targeted by the ASO is at least one of the following. TIFF2025519109000037.tif88161TIFF2025519109000038.tif219161TIFF2025519109000039.tif66161

[0071] In some embodiments, the ASO suppresses uORF 3. In some embodiments, the ASO targets all or part of the sequence of TIFF2025519109000040.tif3128 (SEQ ID NO:, with the start codon highlighted), which includes the 5' part of uORF 3 and several nucleotides up to its 5' end.

[0072] In some embodiments, the sequence of the 5'-UTR that is complementary to the ASO (complementary by about 80% or more, about 85% or more, about 90% or more, about 95% or more, or completely complementary, etc.) and targeted by the ASO is at least one of the following. TIFF2025519109000041.tif81161TIFF2025519109000042.tif219161TIFF2025519109000043.tif73161

[0073] In some embodiments, in an ASO that is "completely complementary" to one of the sequences set forth in SEQ ID NOs: 14 - 157, every nucleobase is involved in the formation of hybridization with the nucleobases of that sequence, and vice versa.

[0074] In some embodiments, the length of the ASO is 10 nucleotides or more, such as 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

[0075] In some embodiments, the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0076] In some embodiments, the ASO is an RNA molecule, a DNA molecule, or a DNA-RNA hybrid molecule. In some embodiments, the ASO contains modified nucleobases, modified linkages, and / or modified sugar moieties.

[0077] In some embodiments, modified sugar moieties include 2'-O-methylated (2'-OMe) modified sugar moieties, locked nucleic acid (LNA) modified sugar moieties, 2'-O-methoxyethyl (2'-MOE) modified sugar moieties, (S)-constrained ethyl nucleic acid (cEt) modified sugar moieties, or 2'-fluoro (2'F) modified sugar moieties.

[0078] In some embodiments, modified linkages include phosphorothioate (PS) linkages, phosphorodiamidate morpholino (PMO) linkages, positively charged PMO linkages, phosphoramidate linkages, methylphosphonate (MP) linkages, phosphorothioate linkages, or peptide nucleic acid (PNA) linkages. In some embodiments, the ASO contains a combination of modified linkages and native phosphodiester (PO) linkages, such as a combination of PS linkages and PO linkages. In some embodiments, the modification may lead to characterizing the ASO as a PMO morpholino.

[0079] In some embodiments, modified nucleobases include 5'-methylcytosine nucleobases, or G-clamp nucleobases.

[0080] In some embodiments, the ASO is conjugated to an N-acetylgalactosamine (GalNAc) group or a cholesterol group. In some embodiments, conjugation improves delivery of the ASO, such as delivery across the cell membrane.

[0081] In some embodiments, the ASO is of the gapmer design. In some embodiments, the gapmer comprises a DNA-based internal "gap" and RNA-like flanking regions. In some embodiments, the RNA-like flanking regions comprise one or more modifications such as 2'-OMe modifications or LNA modifications, among others.

[0082] In some embodiments, the ASO has a nucleotide sequence TIFF2025519109000044.tif4128, a nucleotide sequence TIFF2025519109000045.tif4128, or a variant thereof. The ASO can be RNA-based, DNA-based, or DNA / RNA hybrid-based and can include modifications to the nucleobases, linkages, or sugar moieties, and it is noted that the ASO molecules herein are not intended to be limited by the properties of the molecules as suggested by the recited sequences. For example, the sequence TIFF2025519109000046.tif4128 suggests that this ASO molecule is an RNA molecule, but this sequence is also intended to include the corresponding DNA molecule, the corresponding DNA / RNA hybrid molecule, and the corresponding nucleic acids containing modified nucleobases, modified linkages, and / or modified sugar moieties.

[0083] In some embodiments, the concentration of the ASO in the diseased tissue (kidney or lung) after administration is in the range of about 1 nm to about 100 nm, such as about 2 nm to about 75 nm, about 3 nm to about 50 nm, about 4 nm to about 40 nm, about 5 nm to about 30 nm, or about 10 to about 20 nm.

[0084] In some embodiments, the subject is a mammal such as a human.

[0085] Method for increasing PKD1 / polycystin-1 (PC1) expression in cells In some embodiments, the present disclosure is directed to a method for increasing PKD1 / PC1 expression in cells.

[0086] In some embodiments, the method comprises contacting a cell with an effective amount of a compound that suppresses the first upstream open reading frame (uORF) and / or the second uORF of the PKD1 gene.

[0087] In some embodiments, the cell has a mutation in the PKD1 gene, the PKD2 gene, the PRKCSH gene, the SEC63 gene, the GANAB gene, the ALG8 gene, the ALG9 gene, the SEC61B gene, or the DNAJB11 gene.

[0088] In some embodiments, the cell is in a tissue or a subject.

[0089] In some embodiments, the cell is a kidney cell in a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or a liver cell in a subject diagnosed with polycystic liver disease (PCLD).

[0090] In some embodiments, the compound comprises: a CRISPR component that disrupts the genomic DNA sequence encoding the first uORF and / or the second uORF, or an expression vector expressing the CRISPR component; or an antisense oligonucleotide (ASO) that blocks the first uORF and / or the second uORF, or an expression vector expressing the ASO.

[0091] Such CRISPR components and ASOs are described in detail elsewhere herein.

[0092] In some embodiments, the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, and the CRISPR component disrupts the start codon of the first uORF and / or the second uORF.

[0093] In some embodiments, the compound comprises an ASO or an expression vector expressing the ASO, and the portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, for example within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF or the second uORF.

[0094] In some embodiments, the length of the ASO is 10 nucleotides or more, such as 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

[0095] In some embodiments, the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0096] In some embodiments, the ASO has a nucleotide sequence TIFF2025519109000047.tif4128, the nucleotide sequence TIFF2025519109000048.tif4128, or variants thereof that include modified nucleobases or modified linkages.

[0097] In some embodiments, the modified nucleobases include 2'-O-methylated modified nucleobases.

[0098] In some embodiments, the modified linkages include phosphorothioate (PS) linkages.

[0099] In some embodiments, the concentration of the ASO in contact with the cells ranges from about 1 nm to about 100 nm, such as from about 2 nm to about 75 nm, from about 3 nm to about 50 nm, from about 4 nm to about 40 nm, from about 5 nm to about 30 nm, or from about 10 to about 20 nm.

[0100] Suppression of PKD1 uORF by CRISPR technique In some embodiments, the CRISPR components are designed to induce a targeted genetic modification to the sequence of the uORF in the 5'-UTR of the PKD1 gene, for example, to the start codon ("AUG / ATG") of the uORF in genomic DNA.

[0101] The CRISPR / Cas9 system is a convenient and efficient system for inducing targeted genetic modifications. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a protospacer adjacent motif (PAM) sequence containing conserved dinucleotides upstream of the gRNA binding region. Thereby, the CRISPR / Cas9 system can be designed to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, which makes this system uniquely suitable for editing multiple genes or synergistically activating target genes.

[0102] The Cas9 protein and guide RNA form a complex that identifies and cleaves a target sequence. Cas9 is composed of six domains: REC I, REC II, the bridge helix, PAM interaction, HNH, and RuvC. The RecI domain binds to the guide RNA, while the bridge helix binds to the target DNA. The HNH domain and the RuvC domain are nuclease domains. The guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. When the guide RNA binds to the Cas9 protein, a conformational change occurs and the protein is activated. Once activated, Cas9 searches for the target DNA by binding to a sequence that matches its protospacer adjacent motif (PAM) sequence. The PAM is a base sequence of two or three nucleotides that is within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, the Cas9 protein melts the bases upstream of the PAM and pairs them with the complementary regions on the guide RNA. Then, the RuvC and HNH nuclease domains cut the target DNA behind the third nucleotide base upstream of the PAM.

[0103] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Patent Application Publication No. US2014 / 0068797. CRISPRi uses an RNA-guided Cas9 endonuclease to introduce a DNA double-strand break, thereby inducing a permanent gene disruption where error-prone repair pathways are induced and frameshift mutations occur. Catalytically dead Cas9 has lost its endonuclease activity. When co-expressed with the guide RNA, a DNA recognition complex is created that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently suppresses the expression of the targeted gene.

[0104] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific to the target gene and a Cas endonuclease are introduced into a cell and they form a complex that enables the Cas endonuclease to introduce a double-strand break in the target gene. In certain embodiments, the CRISPR / Cas system includes an expression vector such as, but not limited to, a pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces the expression of the Cas9 endonuclease. Other endonucleases may also be used, including, but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

[0105] In certain embodiments, inducing the Cas expression vector includes exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be recognized that other inducible promoters may also be used. The inducing agent can be a selection condition (e.g., exposure to an agent, such as an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0106] In certain embodiments, the guide RNA and Cas9 can be delivered to the cell as a ribonucleoprotein (RNP) complex. The RNP is composed of purified Cas9 protein complexed with gRNA and is well known in the art to be efficiently delivered to a number of cell types including, but not limited to, stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI).

[0107] The guide RNA is specific to the target genomic region and targets that region for Cas endonuclease-induced double-strand breaks. The target sequence of the guide RNA sequence may be within the locus of a gene or within a non-coding region of the genome. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more nucleotides in length.

[0108] A guide RNA (gRNA), also referred to as a "short guide RNA" or "sgRNA", provides both target specificity and scaffold formation / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a target sequence and a scaffold sequence derived from the endogenous bacterial crRNA and tracrRNA. The gRNA is used to target Cas9 to a designated genomic locus in genomic engineering experiments. The guide RNA can be designed using standard tools well known in the art.

[0109] In the context of CRISPR complex formation, a "target sequence" refers to a sequence that is designed such that the guide RNA has some complementarity thereto, where hybridization between the target sequence and the guide sequence promotes CRISPR complex formation. Complete complementarity is not necessarily required, so long as there is sufficient complementarity to cause hybridization and promote CRISPR complex formation. The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence can be present in an organelle of a eukaryotic cell, such as a mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs). Similar to the target sequence, complete complementarity is not thought to be required so long as the complementarity is sufficient to be functional.

[0110] In certain embodiments, one or more vectors that drive the expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system results in the formation of CRISPR complexes at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors can provide any components of the CRISPR system not included in the first vector. The CRISPR system elements combined in a single vector can be arranged in any suitable orientation, such that one element is located 5' (its "upstream") or 3' (its "downstream") of a second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of a second element, and can be oriented in the same or opposite direction. In certain embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme and one or more of a guide sequence, a tracr-mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0111] In certain embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., in addition to the CRISPR enzyme, about one or more, about two or more, about three or more, about four or more, about five or more, about six or more, about seven or more, about eight or more, about nine or more, about ten or more domains). The CRISPR enzyme fusion protein may include any additional protein sequences, and optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that can form part of a fusion protein containing a CRISPR enzyme are described in U.S. Patent Application Publication No. US20110059502, which is incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0112] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the CRISPR system to cells in culture or to cells in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery media such as liposomes. Viral vector delivery systems include DNA viruses and RNA viruses that have either an episomal genome or an integrated genome after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26).

[0113] In certain embodiments, CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from the Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0114] Generally, Cas proteins contain at least one RNA recognition domain and / or RNA binding domain. The RNA recognition domain and / or RNA binding domain interacts with the guiding RNA. Cas proteins can also include a nuclease domain (i.e., DNase or RNase domain), a DNA binding domain, a helicase domain, an RNAse domain, a protein-protein interaction domain, a dimerization domain, and other domains. Cas proteins can be modified to increase the affinity and / or specificity of nucleic acid binding, modify enzymatic activity, and / or alter other properties of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from the wild-type Cas9 protein or a fragment thereof. In other embodiments, Cas can be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, and the like). Alternatively, domains of the Cas9 protein that are not involved in RNA-guided cleavage can be removed from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Generally, the Cas9 protein contains at least two nuclease (i.e., DNase) domains. For example, the Cas9 protein can contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC domain and the HNH domain cooperate to cut a single strand and create a double-strand break in the DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either the RuvC-like nuclease domain or the HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., has no nuclease activity).In some embodiments where one of the nuclease domains is inactive, the Cas9-derived protein can introduce a nick into double-stranded nucleic acid (such proteins are named "nickase"), but cannot cleave double-stranded DNA. In any of the above embodiments, well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and whole gene synthesis, as well as other methods known in the art, can be used to inactivate any or all of the nuclease domains by one or more deletion mutations, insertion mutations, and / or substitution mutations.

[0115] In one non-limiting embodiment, the vector drives the expression of the CRISPR system. There are abundant suitable vectors useful herein in the art. The vectors used are suitable for replication in eukaryotic cells and optionally for integration. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. The vectors herein can also be used in standard gene delivery protocols for nucleic acids. Methods for gene delivery are known in the art (U.S. Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are hereby incorporated by reference in their entirety).

[0116] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, for example, Sambrook et al. (4 th(Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and are described in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, Sindbis viruses, gammaretroviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0117] In some embodiments, the invention includes any other method for effecting gene knockdown and / or gene editing that enables deletion and / or inactivation of uORFs, such as those described in WO 2018 / 236840, which is hereby incorporated by reference in its entirety.

[0118] Suppression of PKD1 uORF by ASO technology In some embodiments, the ASO molecule is designed to hybridize to the 5'-UTR of PKD1 mRNA at or near the uORF (e.g., at or near the start codon of the uORF), resulting in a substantial reduction in the ribosome binding efficiency of the uORF.

[0119] Antisense oligonucleotides (ASOs) are short strands of nucleotide analogs that hybridize in a sequence-specific manner with complementary mRNA. The technology was first described in the 1970s and has been developed since the 1980s by incorporating advances in oligonucleotide chemistry and formulation. Currently, there are several approved ASO drugs and a significant number of ASO drugs in development. (Bennett, Annual Review of Medicine, Vol. 70:307-321, 2019).

[0120] ASOs are typically 13 - 25 nucleotides in length, but are not limited to this. ASOs are complementary to the mRNA they target and are capable of hybridizing to the mRNA through Watson-Crick base pairing. The sequences of ASOs can be designed with the aid of available software and algorithms. The design of ASOs targeting a specific sequence is described, for example, in Chan et al. (CEPP, Volume33, Issue5-6 May / June 2006, Pages 533-540).

[0121] The formation of the mRNA / ASO duplex can cause two effects: 1) ribonuclease H (RNaseH)-mediated cleavage that degrades the duplex, and 2) steric hindrance that restricts ribosome access to the segment of mRNA complementary to the ASO.

[0122] According to certain embodiments, it is desirable to provide a steric hindrance to restrict access to the uORF in the 5'-UTR of PKD1 mRNA (which increases translation of PC1 protein from PKD1 mRNA) without causing degradation of the mRNA (which reduces expression of PKD1 mRNA and its translation into PC1 protein). Thus, in some embodiments, the ASOs herein include chemical modifications that render the formed RNA / ASO duplex resistant to endonuclease cleavage. Sugar modifications such as 2'-O-methyl (2'-OMe) modification or 2'-O-methoxyethyl (2'-O-MOE) modification, and linkage modifications such as phosphorothioate modification and phosphorothioate (PS) modification are known to increase resistance of the RNA / ASO duplex to endonuclease cleavage (see, for example, Gagliardi et al., Biomedicines. 2021 Apr; 9(4): 433 and Crooke et al., Nucleic Acids Res. 2020 Jun 4; 48(10): 5235-5253). Thus, in some embodiments, the ASOs herein include one or more of such modifications.

[0123] The design of ASOs for improved delivery is also described in Roberts et al. (Nature Reviews Drug Discovery volume 19, pages 673-694 (2020)).

[0124] Vector Vectors can increase the stability of nucleic acids, make delivery easier, or enable expression of nucleic acids or their protein products in cells.

[0125] Thus, in some embodiments, compounds that suppress the PKD1 uORF are incorporated into vectors.

[0126] In some embodiments, the present specification relates to vectors comprising the nucleic acid sequences or constructs of the present specification. The choice of vector depends on the host cell into which it will later be introduced. In certain embodiments, the vectors of the present specification are expression vectors. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic vector and / or eukaryotic vector-based systems can be employed for use herein to produce polynucleotides or their corresponding polypeptides. Many such systems are commercially available and widely accessible.

[0127] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art and is described, for example, in manuals of virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0128] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 families of adeno-associated virus. In some embodiments, the viral vector is a viral vector capable of infecting humans. A desired nucleic acid sequence, such as the above-described compound, can be inserted between the inverted terminal repeats (ITRs) in AAV. In various embodiments, the viral vector is AAV2 or AAV8. The promoter can be the thyroxine-binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desired nucleic acid expression in the liver. The AAV can be a recombinant AAV that originates from an AAV serotype with a capsid and the ITRs originate from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 capsids. In various embodiments, the ITRs in AAV are at least one ITR selected from the group consisting of the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8. In various embodiments, the present specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that causes increased systemic expression of the desired nucleic acid when administered to a subject. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein.

[0129] In some embodiments, the vector into which the nucleic acid sequence is introduced is a plasmid that is either integrated or not integrated into the genome of the host cell when it is introduced into the cell. Exemplary non-limiting examples of vectors into which the nucleotide sequences of the present specification or the gene constructs of the present specification can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0130] Vectors can be obtained by conventional methods that are known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0131] In certain embodiments, the recombinant expression vector may also contain a nucleic acid molecule encoding a peptide inhibitor or peptidomimetic inhibitor of the present disclosure described elsewhere herein.

[0132] A promoter can be one that is naturally associated with a gene or polynucleotide sequence, such as can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer can be one that is naturally associated with a polynucleotide sequence and is located either downstream or upstream of that sequence. Alternatively, certain advantages are obtained by positioning a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers from other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers containing "non-naturally occurring", i.e., different elements of different transcriptional regulatory regions, and / or mutations that modify expression. In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, the sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques including PCR™ in connection with the compositions disclosed herein (U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906). Moreover, it is contemplated that control sequences that direct transcription and / or expression of sequences within subcellular organelles other than the nucleus, such as mitochondria, chloroplasts, etc., can likewise be employed.

[0133] It is important to employ a cell type, organelle, and promoter and / or enhancer that effectively direct the expression of a DNA segment in an organism selected for expression. Those skilled in the art of molecular biology generally understand how to use combinations of promoters, enhancers, and cell types for protein expression. The promoter employed may be constitutive, tissue-specific, inducible, and / or useful for directing high-level expression of the introduced DNA segment under appropriate conditions, which high-level expression is advantageous, for example, for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0134] The recombinant expression vector may also contain a selectable marker gene that facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are proteins such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes encoding immunoglobulins or portions thereof, such as the Fc portion of an immunoglobulin, preferably IgG. The selectable marker may be introduced on a vector separate from the nucleic acid of interest.

[0135] Combination therapy In some embodiments, a method of treating, alleviating, and / or preventing polycystic disease comprises administering to a subject an effective amount of at least one compound and / or composition contemplated within the present disclosure.

[0136] In some embodiments, a composition for treating polycystic disease comprises at least one compound and / or composition contemplated within the present disclosure.

[0137] In some embodiments, at least one additional agent that treats, alleviates, and / or prevents the diseases and / or disorders contemplated herein is further administered to the subject. In other embodiments, the compound and at least one additional agent are co-administered to the subject. In still other embodiments, the compound and at least one additional agent are co-formulated.

[0138] The compounds contemplated within the present disclosure are intended to be useful in combination with one or more additional compounds. These additional compounds can include the compounds of the disclosure of the present invention and / or at least one additional agent for treating polycystic disease, and / or at least one additional agent for treating one or more diseases or disorders contemplated herein.

[0139] The synergistic effect can be calculated, for example, using suitable methods such as the sigmoid-E max Equations (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the Loewe additivity equation (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326), and the median effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55), etc. Each of the above equations can be applied to experimental data to assist in creating the corresponding graph for evaluating the effect of the drug combination. The corresponding graphs associated with the above-mentioned equations are the concentration-effect curve, the isobologram curve, and the combination index curve, respectively.

[0140] Administration / Dosage / Formulation The dosing regimen can affect what constitutes an effective amount. The therapeutic formulations contemplated within the present disclosure may be administered to a subject either before or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, and staggered dosages may be administered daily or sequentially, or the dosage may be administered by continuous infusion, or by bolus injection. Further, the dosage of the therapeutic formulations contemplated within the present disclosure may be proportionally increased or decreased as indicated by the exigencies of the treatment or prevention situation.

[0141] Administration of the compositions contemplated within the present disclosure to a patient, preferably a mammal, more preferably a human, can be effected using known procedures, at an effective dosage and for a period of time effective to treat the diseases and / or disorders contemplated herein in the patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect will vary according to factors such as the condition of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the present disclosure to treat the diseases and / or disorders contemplated herein in the patient. The dosing regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as indicated by the exigencies of the treatment situation. A non-limiting example of an effective dosage range for the therapeutic compounds contemplated within the present disclosure is from about 1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art will be able to study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0142] The actual dosage level of the active ingredient in the pharmaceutical compositions contemplated within the present disclosure may be varied so that an amount of the active ingredient that is effective to achieve the desired therapeutic response is obtained without being toxic to the patient, for a particular patient, composition, and mode of administration.

[0143] In particular, the dosage level to be selected depends on a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, or substances used in combination with the compound, the age, sex, weight, condition, general health, and medical history of the patient to be treated, as well as similar factors well known in the medical arts.

[0144] A physician having ordinary skill in the art, such as an internist or veterinarian, can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, an internist or veterinarian can start with a level of the dosage of the compound contemplated within the present disclosure employed in the pharmaceutical composition that is lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0145] In certain embodiments, it is particularly advantageous to formulate the compounds in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of the therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the present disclosure are determined by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such therapeutic compounds for the treatment of the diseases and / or disorders contemplated herein.

[0146] In certain embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0147] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), their suitable mixtures, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as saccharides, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol. Sustained absorption of the injectable composition can be achieved by including in the composition agents that delay absorption, such as aluminum monostearate or gelatin.

[0148] In certain embodiments, the compositions of the present disclosure are administered to a patient at a dosage range of once to five times or more per day. In another embodiment, the compositions of the present disclosure are administered to a patient at a dosage range including, but not limited to, once a day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the dosing frequency of the various combination compositions of the present disclosure varies among individuals depending on many factors including, but not limited to, age, the disease or disorder being treated, gender, overall health status, and other factors. Therefore, the present disclosure should not be construed as limited to any particular dosing regimen, and the exact dosage and composition to be administered to any patient are determined by the attending physician taking into account all other factors regarding the patient.

[0149] The compounds of the present disclosure for administration can be in the range of about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and can be any and all integer or fractional increments therebetween.

[0150] In some embodiments, the dosage of the compounds of the present disclosure is from about 1 mg to about 2,500 mg. In some embodiments, the dosage of the compounds of the present disclosure used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dosage of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all integer or fractional increments thereof.

[0151] In certain embodiments, the disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of polycystic disease in a patient.

[0152] The formulations may be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any of the preferred modes of administration known in the art, such as intracranial, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration. The pharmaceutical preparations may be sterilized and, if desired, mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances, etc. If desired, these may be combined with other active agents, such as other analgesics.

[0153] Any route of administration of the compositions of the disclosure includes oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual, or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as oral or parenteral, e.g., transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., transvaginal and perivaginal), nasal (intra), and (trans)rectal), intravesical, intralung, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intratracheal, inhalation, and topical administration.

[0154] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the disclosure of the invention are not limited to the specific formulations and compositions described herein.

[0155] Oral administration For oral application, particularly suitable are tablets, dragees, solutions, drops, suppositories, or capsules, caplets, and gelcaps. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions can contain one or more agents selected from the group consisting of inert and non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating agents and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate. Tablets may not be coated, or they may be coated by known techniques for appearance or to delay the release of the active ingredient. Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0156] For oral administration, the compounds of the present disclosure may be in the form of tablets or capsules prepared by conventional methods using pharmaceutically acceptable excipients such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose, or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. The liquid preparations may be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fat); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl p-hydroxybenzoate or propyl p-hydroxybenzoate, or sorbic acid).

[0157] The disclosure of the present invention also includes multilayer tablets comprising a layer providing delayed release of one or more compounds of the present disclosure and a further layer providing immediate release of another medicament. By using a wax / pH-sensitive polymer mix, a gastric-insoluble composition can be obtained in which the active ingredient is encapsulated and its delayed release is ensured.

[0158] Parenteral administration For parenteral administration, the compounds of the present disclosure can be formulated for injection or infusion, e.g., for intravenous, intramuscular, or subcutaneous injection or infusion, or for administration as a bolus dose and / or by continuous infusion. Suspensions, solutions, or emulsions in an oily or aqueous vehicle, optionally containing other formulation agents such as suspending agents, stabilizers, and / or dispersing agents, can be used.

[0159] Additional dosage forms Additional dosage forms of the present disclosure include those described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of the present disclosure also include those described in U.S. Patent Application Publication Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of the present disclosure also include those described in PCT Application Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0160] Controlled release formulations and drug delivery systems In certain embodiments, the formulations of the present disclosure can be, but are not limited to, short-term, rapid clearance, and controlled, e.g., sustained release, delayed release, and pulsatile release formulations.

[0161] The term "sustained release" is used in its conventional sense to refer to a pharmaceutical formulation that provides for the gradual release of a drug over an extended period of time and that, although not necessarily, can result in a substantially constant drug blood level over an extended period of time. The period may be as long as one month or more and should be a longer release than an equivalent dose administered in bolus form.

[0162] In the case of sustained release, the compound can be formulated using a suitable polymer or hydrophobic material that provides the compound with sustained release properties. Thus, the compound for use in the methods of the present disclosure may be administered, for example, by injection, in the form of microparticles, or by implantation, in the form of a wafer or disk.

[0163] In certain embodiments of the present disclosure, the compounds of the present disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0164] The term "delayed release" is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for an initial release of a drug after some delay following drug administration and that, although not necessarily, can include a delay of from about 10 minutes to up to about 12 hours.

[0165] The term "pulsed release" is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for the release of a drug in a manner that produces a pulsed plasma profile of the drug following drug administration.

[0166] The term "immediate release" is used in its conventional sense to refer to a pharmaceutical formulation that provides for the release of a drug immediately following drug administration.

[0167] As used herein, "short term" refers to any period up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 1 hour, up to about 40 minutes, up to about 20 minutes, or up to about 10 minutes following drug administration, and any or all integer or fractional increments thereof.

[0168] As used herein, rapid disappearance refers to any period up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 1 hour, up to about 40 minutes, up to about 20 minutes, or up to about 10 minutes after drug administration, and any and all integer or fractional increments thereof.

[0169] Dosage The therapeutically effective amount or therapeutically effective dosage of the disclosed compounds of the present invention depends on the age, sex, and weight of the patient, the current medical condition of the patient, and the progression of polycystic disease in the patient being treated. Those skilled in the art can determine the appropriate dosage depending on these and other factors.

[0170] Suitable dosages of the disclosed compounds of the present invention can range from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as from about 5 mg to about 250 mg per day. The dosage can be a single dosage or multiple dosages, for example, administered 1 to 4 or more times per day. When using multiple dosages, the amount of each dosage may be the same or different. For example, a dosage of 1 mg per day may be administered as two 0.5 mg dosages with an interval between dosages of about 12 hours.

[0171] It is understood that the amount of compound administered per day may be administered daily, every other day, every two days, every three days, every four days, or every five days in non-limiting examples. For example, in alternate-day administration, a dosage of 5 mg per day may be started on Monday, the first subsequent dosage of 5 mg per day may be administered on Wednesday, and the second subsequent dosage of 5 mg per day may be administered on Friday, and so on.

[0172] If the patient's condition improves, at the discretion of the physician, administration of the modulators of the present disclosure may be given optionally and continuously; alternatively, the dosage of the drug being administered may be temporarily reduced or temporarily discontinued for a specific period (i.e., "drug holiday"). The length of the drug holiday may vary arbitrarily between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dosage reduction during the drug holiday includes 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0173] If improvement occurs in the patient's condition, maintenance dosing may be administered as needed. Subsequently, the dosage or dosing frequency or both are reduced to a level at which the improved disease is maintained as a function of the patient's condition. In certain embodiments, upon recurrence of any symptoms and / or infections, the patient will require long-term intermittent treatment.

[0174] The compounds for use in the methods of the present disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage amounts for the patient being treated, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or for one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0175] The toxicity and therapeutic efficacy of such treatment regimens are optionally determined in cell cultures or experimental animals, which includes LD 50 (the dose lethal to 50% of the population) and ED50 (Effective Therapeutic Dose in 50% of the Population) including, but not limited to, the determination thereof. The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio between LD 50 and ED 50 . Capsid assembly modulators exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used optionally in formulating dosage ranges for use in humans. The dosage of such a capsid assembly modulator preferably lies within the range of circulating concentrations that include the ED 50 with minimal toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized.

[0176] One of ordinary skill in the art can recognize or confirm numerous equivalents to the specific procedures, aspects, claims, and examples described herein using only routine experimentation. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims appended hereto. For example, modifications in assays and / or reaction conditions using alternative methods recognized in the art and using only routine experimentation are understood to be within the scope of this application.

Examples

[0177] This specification is described in more detail below with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting, unless otherwise specifically designated. Therefore, this specification should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0178] Example 1: Overview A substantial subset of PCLD and ADPKD is caused by insufficient functional gene dosage of PC1, which is caused by either a loss-of-function mutation in the PKD1 gene itself or a mutation in an ER gene responsible for the maturation of PC1 from the endoplasmic reticulum (ER) to the cell surface of the primary cilium. Thus, it is expected that increasing the functional gene dosage of PC1 may be able to treat, alleviate, or prevent PCLD and ADPKD.

[0179] As described in Example 1, in the study described herein (the "present study"), two upstream open reading frames (uORFs) were discovered in the 5'-untranslated region (5'-UTR) of PKD1 messenger RNA (mRNA). Further in the present study, it was discovered that the two uORFs reduce the translation efficiency of PKD1, and that translation of PKD1 mRNA is significantly enhanced and the protein level of PC1 is increased either by mutating to eliminate the uORFs or by blocking the uORFs using antisense oligonucleotides (ASOs).

[0180] Specifically, in vitro data described herein demonstrated that when the PKD1 5'UTR has an edit of just 2 nucleotides to abolish translation of the uORF, the steady-state expression of the downstream gene increases approximately 4-fold. The present study also shows that inhibition of PKD1 uORF translation in human cells using antisense oligonucleotides (ASOs) nearly recapitulates the fold change in expression in the base editing assessment, and thus this highly specific therapy is also highly achievable.

[0181] The two uORFs in the 5'-UTR of PKD1 have unique sequences, and since methods targeting uORF sequences such as CRISPR or ASO can be designed with extremely high specificity, treatment of ADPKD or PCLD through uORF is expected to be highly specific. Considering the fold change in PKD1 protein expression achieved by inhibiting uORF, a hypothesis is put forward that the magnitude of this effect will be sufficient to prevent cyst formation in at least some models or patients and will be a safe approach for long-term therapy. Also, this method is expected to be effective for all patients with isolated polycystic liver (the symptomatic incidence rate is about 1:10,000 - 1:150,000) and a subset of patients with ADPKD (the overall incidence rate is about 1:400 - 1:1000).

[0182] Example 1-1: Related Information Autosomal dominant polycystic kidney disease (ADPKD) affects 1 in 400 to 1 in 1000 people and causes 5 - 10% of kidney failure. ADPKD results from pathogenic variants in the PKD1 or PKD2 genes that encode polycystin 1 (PC1) and polycystin 2 (PC2), and is characterized by the enlargement of renal and hepatic cysts. ADPKD - ADPLD (also known as isolated polycystic liver or PCLD), which is an overlapping spectrum of cystic phenotypes in the kidney and liver, is caused by mutations in genes required for PC1 maturation in the endoplasmic reticulum (ER): SEC63, PRKCSH, ALG8, ALG9, GANAB, SEC61B, DNAJB117. PKD1 truncating mutations that result in a complete loss of PC1 function lead to the most rapid progression to kidney failure. Patients with non - truncating PKD1 mutations still progress to kidney failure, and in these and many ADPKD - ADPLD cases, there is an urgent need for therapies to prevent the symptoms and complications due to the continuous accumulation and enlargement of renal and / or hepatic cysts. The late onset of kidney failure in those with non - truncating PKD1 mutations, which account for approximately one - third of ADPKD, suggests that the encoded PC1 functions at least partially. A rather large subset of these missense mutant PC1 constructs mature inefficiently to the cell surface, suggesting that quantitative expression is essential. Interestingly, in a cystic kidney mouse model with impaired ER maturation (Prkcsh or Sec63), the quantitative increase in PC1 production achieved by mice carrying a bacterial artificial chromosome encoding three copies of the Pkd1 gene was sufficient to reach the cell surface with enough PC1 to avoid cyst formation. Collectively, these findings suggest that increasing PC1 expression can successfully increase the functional amount reaching the cell surface to an extent that can prevent cyst formation, despite inefficient PC1 maturation.

[0183] Research at the University of Texas Southwestern has suggested an approach to inhibit microRNAs that degrade PKD1 mRNA. In fact, gene mouse models have shown the benefits resulting from increased PC1 expression from this approach (Lakhia et al. Nat Comm. 2022).

[0184] However, since the microRNA approach will target a vast number of genes, therapies based on this will be significantly limited by off-target effects. For this reason, the specificity of targeting the unique sequence of the PKD1 uORF, combined with an expected increase in PC1 protein expression of the same or greater magnitude, is thought to have similar or greater desirable efficacy without risk.

[0185] Examples 1-2: PKD1 mRNA contains at least three upstream open reading frames (uORFs) in the 5'UTR Three potential uORFs in the PKD1 5'-untranslated region (5'UTR) were identified among the top 0.1% of all genome-wide uORFs scored in a computationally determined score of uORF translation potential. The sequences of these uORFs (uORF1, uORF2, uORF3) and the proposed uORF4 are set forth in SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, respectively. Two uORFs (uORF1 and uORF2) starting with ATG are shown in Figure 2A. In addition to the uORFs, the predicted secondary structure of the 5'UTR is further illustrated in Figure 2B.

[0186] The 5' untranslated regions (5'UTRs) of messenger RNA (mRNA) sequences from many genes contain potential translation start sites that encode short peptides or out-of-frame peptides from the intended gene; these are known as upstream open reading frames (uORFs). For some uORFs studied in other genes, translation of the uORF has a detrimental effect on the translation of the intended protein by reducing the number of ribosomes that reach the start codon of the protein sequence (Figures 3A - 3B).

[0187] Examples 1 - 3: Genetically eliminating the uORF in the PKD1 5'UTR significantly increases protein expression Referring to Figures 4A - 4D, constructs were prepared by replacing the 5'UTR of Renilla luciferase in a dual luciferase reporter (Figure 4A) with either the wild-type human PKD1 5'UTR ("wt") or a human PKD1 5'UTR containing one or two single base edits to eliminate one or both of the uORF start sites ("ΔuORF1", "ΔuORF2", and "ΔuORF1&2") (Figure 4B). Cells were then transfected with the four constructs. The mutations that eliminated the uORF did not change mRNA expression or transfection efficiency (Figure 4D). However, this targeted editing resulted in a nearly 4-fold significant increase in the expression of the luciferase protein (Figure 4C, "wt" vs. "1&2").

[0188] Examples 1 - 4: ASOs that inhibit the uORF in the PKD1 5'UTR significantly increase protein expression Based on the sequences of the uORF and the 5'UTR, two non-limiting examples of antisense oligonucleotides: uORF1 ASO ("ASO1") TIFF2025519109000049.tif4128located before and including the AUG start codon of uORF1, complementary to the 5'UTR sequence), and uORF2 ASO ("ASO2") We designed two antisense oligonucleotides (ASOs) (complementary to the 5'UTR sequence that is located upstream of and includes the AUG start codon of 4128uORF2 of TIFF2025519109000050). Both ASOs contain 2'-O-methylated modified nucleobases and phosphorothioate (PS) linkages to increase resistance to degradation.

[0189] After transfecting cells that express a luciferase reporter containing the wild-type 5'UTR of PKD1 (see the section "Examples 1-3"), the cells were incubated with either one or both of the ASOs (ASO1, ASO2, or ASO1+2), or mismatch control (MM1, MM2) ASOs. Lysates were collected after 24 hours of treatment. Referring to Figure 5, luciferase expression in the lysates of cells treated with both the uORF1 ASO and the uORF2 ASO showed an approximately 3-fold increase compared to the lysates of cells treated with the control.

[0190] Referring to Figure 6A, when wild-type epithelial cells were treated with 20 nM of the ASO, a 1.5- to 2-fold increase in the steady-state expression level of PC1 was observed compared to that seen in the mismatch ASO, without any significant change in mRNA expression. This is consistent with the proposed mechanism of uORF inhibition that promotes translation without acting on the transcript. The increase in PC1 expression can be observed with 12, 24, 48, and 96 hours of treatment (Figures 6B-6D). Referring to Figure 6E, a strong upregulation of PC1 protein expression was observed when the concentration of the ASO was 10 nM or 20 nM.

[0191] Example 1-5: Design of ASOs for sterically blocking the translation of mouse Pkd1 uORF Referring to Figure 8, examination of the mouse Pkd1 mRNA sequence reveals two potential uORFs (mouse uORF1 and mouse uORF2) that start with ATG and correspond to the two human PKD1 uORFs.

[0192] Referring to FIG. 9, two antisense oligonucleotides (mouse ASO1 and mouse ASO2) for suppressing the mouse uORF, as well as two mismatch control antisense oligonucleotides (mouse MM-1 and mouse MM-2), were designed with reference to the publicly available information (Liang et al. Nat Biotechnol 34, 875-880 (2016) and Liang et al. (Nucleic acids research 45, 9528-9546 (2017)). Specifically, antisense oligonucleotides each having a length of 16 nucleotides and containing 2'-O-methyl (2'-OMe) modification and phosphorothioate (PS) linkage were designed to have the following sequences: mouse uORF-1 ASO (mouse ASO1): TIFF2025519109000051.tif11164, mouse mismatch control 1 (mouse MM-1): TIFF2025519109000052.tif11164, mouse uORF-2 ASO (mouse ASO2): TIFF2025519109000053.tif11162, and mouse mismatch control 2 (mouse MM-2): TIFF2025519109000054.tif11162. In the antisense oligonucleotides of this paragraph, "m" indicates that these antisense oligonucleotides have a 2'-O-methyl (2'-OMe) modification on the sugar group, and "*" indicates that the linkage is a phosphorothioate (PS) linkage.

[0193] The two mouse ASOs and two control ASOs were tested in a mouse cell line (containing an HA epitope tag on the C-terminus of Pkd1 to enable assessment of the C-terminal fragment). Referring to FIGS. 10A and 10B, when the mouse cell line was treated with 20 nM of mouse ASO1, ASO2, or a combination of the two ASOs, the level of mature PC1 protein in the cell line increased significantly.

[0194] Next, ASO was tested in another mouse cell line that contains the missense mutation R2216W in the PC1 protein and a V5 epitope tag for detection on the C-terminus of the same protein. The mouse R2216W mutation corresponds to the R2220W mutation in the human PC1 protein found in some ADPKD patients and has been shown to affect protein cleavage and maturation (Vujic et al. J Am Soc Nephrol. 2010 Jul;21(7):1097-102, and Krappitz et al JASN 2023). Referring to Figure 11, treatment with ASO resulted in a significantly increased level of mature PC1 protein (cleaved protein), even for the mutant PC1 R2216W protein that tends to be resistant to cleavage and maturation.

[0195] Example 2: (Hypothesis) ASOs that suppress uORF3 and / or uORF4 increase PC1 protein levels In addition to the above uORF1 and uORF2, further analysis of the human PKD1 mRNA 5'-UTR sequence revealed two additional potential uORFs, namely uORF3 and uORF4, with evidence of translation in at least one publicly available ribosome profiling experiment (Figure 12).

[0196] Design ASOs to suppress uORF3 and uORF4. Specifically, design the ASOs to bind to the human PKD1 mRNA molecule either near or including the start codon of uORF3 or uORF4 (both uORF3 and uORF4 have "CUG" as the start codon) and to prevent translation of either or both of these two uORFs. Testing of the ASOs (such as in human cell lines) indicates that these ASOs are capable of increasing the expression of PC1 protein and its mature form, similar to ASO1 / 2 that suppress uORF1 / 2.

[0197] Example 3: (Hypothesis) An ASO that inhibits the uORF in the PKD1 5'UTR reduces the cystic phenotype in an ADPKD mouse model Referring to FIG. 7, knocking out Dnajb11 and one copy of Pkd1 during embryonic development (Ksp-Cre is active in the distal nephron from mid-gestation) results in a strong cystic phenotype in mice.

[0198] Using this mouse disease model, it has been found that administration of the mouse uORF ASO significantly increases PC1 protein expression from the remaining single allele, with a two-fold increase, and it is expected that the cystic phenotype of the mouse kidneys will disappear.

[0199] Example 4: (Hypothesis) Evaluation of uORF starting with CTG by Western blot Cultured retinal pigment epithelial (RPE) cells are applied with either vehicle, ASO3 (targeting uORF3), a mismatch ASO3 that is a negative control ("ASOMM3", a modified sequence that does not actually target uORF3), ASO1 (positive control), or ASO1 + ASO3 for 24 hours or 96 hours to create protein lysates from the cells and evaluate polycystin-1 (PC1) expression by Western blot.

[0200] The experiment is repeated to compare the normalized luciferase expression from wild-type PKD1 5'UTR - Renilla luciferase constructs treated with vehicle, ASO3, ASOMM3, ASO1, or ASO1 + ASO3.

[0201] The effect of blocking uORF3 on PC1 expression will be similar to the effect of blocking the other uORFs described herein.

[0202] In further experiments, the sequence of PKD1-5'UTR in our psiCHECK2 luciferase expression plasmid containing the human PKD1 5'UTR is modified to contain the gene sequence modification ΔuORF3, or ΔuORF4, or a combination of these with ΔuORF1 or ΔuORF2 similar to those illustrated in FIG. 4B. Modification of the gene sequence changes the CTG start codon of uORF3 or uORF4 to TTG, preventing translation initiation. Renilla:firefly luciferase expression from the gene-modified construct compared to Renilla:firefly luciferase expression from the wild-type construct (baseline) and Renilla:firefly luciferase expression from the delta-uORF1 construct (positive control based on the illustrated results).

[0203] Enumerated embodiments In some embodiments, this specification is directed to the following non-limiting embodiments: Embodiment 1: A method of treating, alleviating, and / or preventing it in a subject in need thereof for the treatment, alleviation, and / or prevention of autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD), comprising: administering to the subject an effective amount of a compound that suppresses translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene comprising the method. Embodiment 2: A method of treating, alleviating, and / or preventing ADPKD in a subject, wherein the ADPKD is caused by or associated with a mutation in the PKD1 gene in the subject, according to the method of Embodiment 1. Embodiment 3: A method of treating, alleviating, and / or preventing PCLD in a subject, wherein the PCLD is caused by or associated with a germline mutation in the PKD1 gene, PKD2 gene, PRKCSH gene, SEC63 gene, GANAB gene, ALG8 gene, ALG9 gene, SEC61B gene, or DNAJB11 gene in the subject, according to the method of Embodiment 1. Aspect 4: The compound is as follows: A CRISPR component that disrupts the genomic DNA sequence encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the CRISPR component; or An antisense oligonucleotide (ASO) that blocks the translation of the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the ASO The method according to any one of Aspects 1 to 3, comprising the above. Aspect 5: The method according to Aspect 4, wherein the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, and the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF. Aspect 6: The method according to Aspect 4, wherein the compound comprises an ASO or an expression vector expressing the ASO, and the portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, for example within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF. Aspect 7: The method according to Aspect 4 or 6, wherein the length of the ASO is 10 nucleotides or more, for example 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more. Aspect 8: The methods of Aspects 4 and 6 - 7, wherein the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less. Aspect 9: At least one of the following: (a) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 14 - 61, (b) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 62 - 109, (c) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 110 - 157, (d) The ASO is the nucleotide sequence TIFF2025519109000055.tif4128, the nucleotide sequence TIFF2025519109000056.tif4128, and the methods of Aspects 4 and 6 - 8 apply. Aspect 10: The methods of Aspects 4 and 6 - 9, wherein the ASO comprises a modified nucleobase, a modified sugar moiety, or a modified linkage. Aspect 11: At least one of the following: (a) The ASO comprises a modified sugar moiety, and the modified sugar moiety comprises a 2'-O-methylated modified sugar moiety such as a 2'-O-methylated ribose group, (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage and the method of Aspect 10 applies. Aspect 12: The methods of Aspects 1 - 11, wherein the subject is a mammal such as a human. Aspect 13: The methods of Aspects 1 - 12, wherein the compound comprises an ASO or an expression vector expressing the ASO, and the concentration of the ASO in the kidney or lung of the subject is in the range of about 1 nm to about 100 nm. Aspect 14: A method for increasing the expression of PKD1 in a cell, contacting the cell with an effective amount of a compound that suppresses translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene A method comprising the steps of: Aspect 15: The method of aspect 14, wherein the cell has a mutation in the PKD1 gene, the PKD2 gene, the PRKCSH gene, the SEC63 gene, the GANAB gene, the ALG8 gene, the ALG9 gene, the SEC61B gene, or the DNAJB11 gene. Aspect 16: The method of aspects 14-15, wherein the cell is in a tissue or a subject. Aspect 17: The method of aspects 14-16, wherein the cell is a renal cell in a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or a hepatocyte in a subject diagnosed with polycystic liver disease (PCLD). Aspect 18: The compound is selected from the following: CRISPR components that disrupt the genomic DNA sequence encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the CRISPR components; or Antisense oligonucleotides (ASOs) that block the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector expressing the ASOs The method of aspects 14-17, comprising the above. Aspect 19: The method of aspect 18, wherein the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, and the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF. Aspect 20: The method of embodiment 18, wherein the compound comprises an ASO or an expression vector expressing the ASO, and a portion of the PKD1 mRNA complementary to the ASO extends within 5 nucleotides, such as within 4 nucleotides, within 3 nucleotides, within 2 nucleotides, within 1 nucleotide, from the start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF, reaches the boundary of the start codon, reaches 1 or more nucleotides of the start codon, reaches 2 or more nucleotides of the start codon, or reaches the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF. Embodiment 21: The method of embodiment 18 or embodiment 20, wherein the length of the ASO is 10 nucleotides or more, such as 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more. Embodiment 22: The methods of embodiments 18 and 20 - 21, wherein the length of the ASO is 30 nucleotides or less, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less. Embodiment 23: At least one of the following: (a) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 14 - 61; (b) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 62 - 109; (c) The ASO is completely complementary to one of the sequences set forth in SEQ ID NOs: 110 - 157; (d) The ASO has a nucleotide sequence TIFF2025519109000057.tif4128, and includes the nucleotide sequence TIFF2025519109000058.tif4128 applies to the methods of embodiments 18 and 20 - 22. Embodiment 24: The methods of embodiments 18 and 20-23, wherein the ASO comprises a modified nucleobase, a modified sugar, or a modified linkage. Embodiment 25: At least one of the following: (a) The ASO comprises a modified sugar, and the modified sugar comprises a 2'-O-methylated modified sugar group such as a 2'-O-methylated ribose group; (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage applies to the method of embodiment 24. Embodiment 26: The methods of embodiments 14-24, wherein the compound comprises an ASO or an expression vector expressing the ASO, and the concentration of the ASO contacted with the cells ranges from about 1 nM to about 100 nM.

[0204] The foregoing outlines the characteristics of several embodiments so that those skilled in the art can better understand aspects of the disclosure of the present invention. Those skilled in the art should recognize that they can readily use the disclosure of the present invention as a basis for designing or modifying other processes and structures to perform the same purposes and / or achieve the same advantages as the embodiments introduced herein. Those skilled in the art should also understand that such equivalent constructs do not depart from the spirit and scope of the disclosure of the present invention, and that various changes, substitutions, and modifications can be made by those skilled in the art without departing from the spirit and scope of the disclosure of the present invention.

Claims

1. A method for treating, alleviating, and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in subjects requiring treatment, alleviation, and / or prevention of such disease, The process of administering to a subject an effective amount of a compound that suppresses the translation of the first upstream open reading frame (uORF), second uORF, third uORF, and / or fourth uORF of the PKD1 gene. Methods that include...

2. A method for treating, mitigating, and / or preventing ADPKD in a subject, wherein the ADPKD is caused by or associated with a mutation in the PKD1 gene in the subject.

3. A method for treating, mitigating, and / or preventing PCLD in a subject, wherein the PCLD is caused by or associated with germline mutations in the PKD1, PKD2, PRKCSH, SEC63, GANAB, ALG8, ALG9, SEC61B, or DNAJB11 gene in the subject.

4. The aforementioned compound is as follows: A CRISPR component that disrupts the genomic DNA sequences encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector that expresses said CRISPR component; or Antisense oligonucleotides (ASOs) that block the translation of the first uORF, second uORF, third uORF, and / or fourth uORF, or an expression vector expressing said ASO. A method according to any one of claims 1 to 3, including the method described in any one of claims 1 to 3.

5. The method according to claim 4, wherein the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, the CRISPR component disrupts the start codons of a first uORF, a second uORF, a third uORF, and / or a fourth uORF.

6. The method according to claim 4, wherein the compound comprises an ASO or an expression vector expressing the ASO, the portion of PKD1 mRNA complementary to the ASO extends within 5 nucleotides away from the start codon of a first uORF, a second uORF, a third uORF, or a fourth uORF, for example, extending within 4 nucleotides, extending within 3 nucleotides, extending within 2 nucleotides, extending within 1 nucleotide, reaching the boundary of the start codon, reaching 1 or more nucleotides of the start codon, reaching 2 or more nucleotides of the start codon, or reaching the entire start codon of the first uORF, a second uORF, a third uORF, or a fourth uORF.

7. The method according to claim 4, wherein the length of the ASO is 10 nucleotides or more, for example, 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

8. The method according to claim 4, wherein the length of the ASO is 30 nucleotides or less, for example, 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

9. At least one of the following: (a) ASO is completely complementary to one of the sequences described in SEQ ID NO: 14-61. (b) ASO is completely complementary to one of the sequences described in SEQ ID NO: 62–109. (c) ASO is perfectly complementary to one of the sequences described in SEQ ID NO: 110-157. (d) The ASO contains the nucleotide sequences CAUGGCGGGCGCGGGG (SEQ ID NO: 158) and CAUGGCCCCGCCGUCC (SEQ ID NO: 159). The method described in claim 4, which applies to the above.

10. The method according to claim 4, wherein the ASO comprises a modified nucleic acid base, a modified sugar group, or a modified linkage.

11. At least one of the following: (a) ASO contains a modified sugar group, and the modified sugar group contains a 2'-O-methylated sugar group such as a 2'-O-methylated ribose group, (b) The ASO includes a modified linkage, and the modified linkage includes a phosphorothioate (PS) linkage. The method according to claim 10, which applies to the above.

12. The method according to any one of claims 1 to 3, wherein the subject is a mammal such as a human.

13. The method according to any one of claims 1 to 3, wherein the compound comprises ASO or an expression vector expressing said ASO, and the concentration of said ASO in the target kidney or lung is in the range of about 1 nm to about 100 nm.

14. A method for increasing PKD1 expression in cells, The step of contacting the cells with an effective amount of a compound that suppresses the translation of the first upstream open reading frame (uORF), second uORF, third uORF, and / or fourth uORF of the PKD1 gene. Methods that include...

15. The method according to claim 14, wherein the cells have mutations in the PKD1 gene, PKD2 gene, PRKCSH gene, SEC63 gene, GANAB gene, ALG8 gene, ALG9 gene, SEC61B gene, or DNAJB11 gene.

16. The method according to any one of claims 14 to 15, wherein the cells are present in a tissue or object.

17. The method according to any one of claims 14 to 15, wherein the cells are renal cells from a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or hepatocytes from a subject diagnosed with polycystic liver disease (PCLD).

18. The aforementioned compound is as follows: A CRISPR component that disrupts the genomic DNA sequences encoding the first uORF, the second uORF, the third uORF, and / or the fourth uORF, or an expression vector that expresses said CRISPR component; or Antisense oligonucleotides (ASOs) that block the first, second, third, and / or fourth uORFs, or an expression vector expressing said ASOs. The method according to any one of claims 14 to 15, including the method described in any one of claims 14 to 15.

19. The method according to claim 18, wherein the compound comprises a CRISPR component or an expression vector expressing the CRISPR component, the CRISPR component disrupts the start codons of a first uORF, a second uORF, a third uORF, and / or a fourth uORF.

20. The method according to claim 18, wherein the compound comprises an ASO or an expression vector expressing the ASO, the portion of PKD1 mRNA complementary to the ASO extends within 5 nucleotides away from the start codon of a first uORF, a second uORF, a third uORF, or a fourth uORF, for example, extending within 4 nucleotides, extending within 3 nucleotides, extending within 2 nucleotides, extending within 1 nucleotide, reaching the boundary of the start codon, reaching 1 or more nucleotides of the start codon, reaching 2 or more nucleotides of the start codon, or reaching the entire start codon of the first uORF, a second uORF, a third uORF, or a fourth uORF.

21. The method according to claim 18, wherein the length of the ASO is 10 nucleotides or more, for example, 11 nucleotides or more, 12 nucleotides or more, 13 nucleotides or more, 14 nucleotides or more, or 15 nucleotides or more.

22. The method according to claim 18, wherein the length of the ASO is 30 nucleotides or less, for example, 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

23. At least one of the following: (a) ASO is completely complementary to one of the sequences described in SEQ ID NO: 14-61. (b) ASO is completely complementary to one of the sequences described in SEQ ID NO: 62–109. (c) ASO is perfectly complementary to one of the sequences described in SEQ ID NO: 110-157. (d) The ASO contains the nucleotide sequences CAUGGCGGGCGCGGGG (SEQ ID NO: 158) and CAUGGCCCCGCCGUCC (SEQ ID NO: 159). The method according to claim 18, which applies to the above.

24. The method according to claim 18, wherein the ASO comprises a modified nucleic acid base, a modified sugar group, or a modified linkage.

25. At least one of the following: (a) ASO contains a modified sugar, and the modified sugar group contains a 2'-O-methylated sugar group such as a 2'-O-methylated ribose group, (b) The ASO includes a modified linkage, and the modified linkage includes a phosphorothioate (PS) linkage. The method according to claim 24, which applies to the above.

26. The method according to claims 14 to 15, wherein the compound comprises an ASO or an expression vector expressing the ASO, and the concentration of the ASO that comes into contact with the cell is in the range of about 1 nm to about 100 nm.

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