Methods of treating, ameliorating and / or preventing polycystic kidney disease and polycystic liver disease

Abstract

Described herein is a method of ameliorating 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 inhibits translation of a first upstream open reading frame (uORF), a second uORF, a third uORF, and / or a fourth uORF of the PKD1 gene.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S. Provisional Patent Application No. 63 / 345,634 filed on May 25, 2022 and U.S. Provisional Patent Application No. 63 / 359,109 filed on July 7, 2022, all of which are incorporated herein by reference in their entirety.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant 1K08DK119642-01 from the National Institutes of Health. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] An ASCII text file named "047162-7395WO1(02006)_Seq Listing.xml" created on May 24, 2023, comprising 173 kilobytes, the entire contents of which are hereby incorporated by reference into this document. Background Art

[0007] Autosomal dominant polycystic kidney disease (ADPKD) is a highly penetrant inherited polycystic kidney disease that usually causes cysts and kidney deformation over decades and eventually leads to kidney failure, with most patients requiring dialysis or transplantation after age 50.

[0008] Although ADPKD is sometimes considered an orphan disease, the number of ADPKD patients is actually enormous. It is estimated that there are more than 600,000 people with ADPKD in the United States alone and more than 12 million people worldwide. In addition, because ADPKD is not a basic mutation, but a new (de novo) mutation that occurs all the time, the number of ADPKD patients is expected to increase further as the world's population grows.

[0009] Currently, there is only one approved drug for the treatment of ADPKD, tolvaptan. Unfortunately, tolvaptan has significant side effects. The drug causes polyuria (6.0 ± 1.8 L of urine per day, Kramers et al., BMC Nephrol. 2018;19:157), carries a black box warning for liver damage (the most stringent warning the FDA has for a drug on the market), and is undergoing a risk evaluation and mitigation strategy (REMS) from the FDA. Given that many ADPKD patients require long-term treatment, tolvaptan's significant side effects are particularly unwelcome. In addition, even when tolerated at the maximum therapeutic dose, tolvaptan treatment has little effect on delaying renal failure.

[0010] More than 90% of people with ADPKD also have multiple cysts in their liver. This is called polycystic liver disease. These cysts develop in a similar way to those that affect the bile duct epithelium and kidney tubule epithelium. While liver cysts do not usually lead to liver failure, some patients may experience debilitating symptoms from a severely enlarged liver, including pain, infection, swelling, and early satiety with an inability to maintain adequate nutrition. There are currently no FDA-approved drug therapies for liver cysts. Instead, patients undergo repeated interventions.

[0011] or surgery to aspirate, fenestrate, or remove the cysts, or to perform a partial or total hepatectomy combined with a liver transplant. Clinically indistinguishable polycystic liver disease can occur in the absence of renal cysts when caused by a different but related genetic mechanism. This isolated polycystic liver disease (PCLD, also known as "autosomal dominant polycystic liver disease" or "ADPLD") is considered a rare genetic disease, but its prevalence may approach that of ADPKD if asymptomatic cases identified at autopsy are included. Approximately 20% of patients with PCLD experience obvious clinical symptoms such as dyspnea, early satiety, abdominal distension, malnutrition, gastroesophageal reflux, and back pain, which are caused by hepatomegaly compressing surrounding organs or complications of cysts. Currently, the only radical treatment for PCLD is liver transplantation, which is only used in the most severe cases.

[0012] Therefore, there is a need for new therapeutic approaches to treat polycystic kidney disease and polycystic liver disease. The present invention addresses this need. Summary of the invention

[0013] In some aspects, the present invention relates to the following aspects:

[0014] In one aspect, the present invention provides a method for treating, improving and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof, comprising: administering to the subject an effective amount of a compound that inhibits 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.

[0015] In various embodiments, the method is a method for treating, ameliorating and / or preventing ADPKD in a subject, and wherein the ADPKD is caused by or involves a mutation in the subject's PKD1 gene.

[0016] In various embodiments, the method is a method for treating, ameliorating and / or preventing PCLD in a subject, and wherein PCLD is caused by or involves a germline mutation in the subject's PKD1 gene, PKD2 gene, PRKCSH gene, SEC63 gene, GANAB gene, ALG8 gene, ALG9 gene, SEC61B gene or DNAJB11 gene.

[0017] In various embodiments, the compound comprises:

[0018] 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 that expresses the CRISPR component; or

[0019] 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.

[0020] In various embodiments, the compound includes a CRISPR component or an expression vector expressing a CRISPR component, and wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF.

[0021] In various embodiments, the compound includes an ASO or an expression vector expressing an ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less from the start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF, such as extending to 4 nucleotides or less, extending to 3 nucleotides or less, extending to 2 nucleotides or less, extending to 1 nucleotide or less, reaching the boundary of the start codon, reaching 1 nucleotide or more of the start codon, reaching 2 nucleotides or more of the start codon, or reaching the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0022] In various embodiments, the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer, or 15 nucleotides or longer.

[0023] In various embodiments, the ASO is 30 nucleotides or less in length, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0024] In various embodiments, at least one of the following applies:

[0025] (a) the ASO is completely complementary to a sequence listed in SEQ ID NO: 14-61,

[0026] (b) the ASO is completely complementary to one of the sequences listed in SEQ ID NOs: 62-109,

[0027] (c) the ASO is completely complementary to one of the sequences listed in SEQ ID NOs: 110-157,

[0028] (d) ASO comprises the nucleotide sequence CAUGGCGGGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID ID: 159).

[0029] In various embodiments, the ASO includes a modified base, a modified sugar group, or a modified linkage.

[0030] In various embodiments, at least one of the following applies:

[0031] (a) the ASO comprises a modified sugar group, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group,

[0032] (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

[0033] In various embodiments, the subject is a mammal, such as a human.

[0034] In various embodiments, the compound comprises an ASO or an expression vector expressing an ASO, and wherein the concentration of the ASO in the kidney or lung of the subject ranges from about 1 nm to about 100 nm.

[0035] In another aspect, the present invention provides a method for increasing PKD1 expression in a cell, comprising:

[0036] An effective amount of a compound that inhibits translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene is contacted with the cell.

[0037] In various 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.

[0038] In various embodiments, the cell is located in a tissue or a subject.

[0039] In various 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).

[0040] In various embodiments, the compound comprises:

[0041] 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 that expresses the CRISPR component; or

[0042] 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.

[0043] In various embodiments, the compound includes a CRISPR component or an expression vector expressing a CRISPR component, and wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF, and / or the fourth uORF.

[0044] In various embodiments, the compound includes an ASO or an expression vector expressing an ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less from the start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF, such as extending to 4 nucleotides or less, extending to 3 nucleotides or less, extending to 2 nucleotides or less, extending to 1 nucleotide or less, reaching the boundary of the start codon, reaching 1 nucleotide or more of the start codon, reaching 2 nucleotides or more of the start codon, or reaching the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0045] In various embodiments, the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer, or 15 nucleotides or longer.

[0046] In various embodiments, the ASO is 30 nucleotides or less in length, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0047] In various embodiments, at least one of the following applies:

[0048] (a) the ASO is completely complementary to a sequence listed in SEQ ID NO: 14-61,

[0049] (b) the ASO is completely complementary to one of the sequences listed in SEQ ID NOs: 62-109,

[0050] (c) the ASO is completely complementary to one of the sequences listed in SEQ ID NOs: 110-157,

[0051] (d) ASO comprises the nucleotide sequence CAUGGCGGGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID ID: 159).

[0052] In various embodiments, the ASO includes a modified base, a modified sugar group, or a modified linkage.

[0053] In various embodiments, at least one of the following applies:

[0054] (a) the ASO comprises a modified sugar, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group,

[0055] (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

[0056] In various embodiments, the compound comprises an ASO or an expression vector expressing an ASO, and wherein the concentration of the ASO contacted with the cell ranges from about 1 nm to about 100 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The following detailed description of the exemplary embodiments will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, non-limiting embodiments are shown in the accompanying drawings. However, it should be understood that the present description is not limited to the precise arrangements and tools of the embodiments shown in the accompanying drawings.

[0058] Figures 1A-1C It is shown that reduced functional dosage of PKD1 / polycystin-1 (PC1), either due to loss-of-function mutations in the PKD1 gene itself or due to mutations in endoplasmic reticulum (ER) genes responsible for PC1 maturation, leads to renal cysts, which, according to some embodiments, can be rescued by increasing PKD1 copy number. Figure 1A-1B : Carrying non-truncating mutations in PKD1 (kidney and liver) ( Figure 1A ) or SEC63 mutation (liver only) ( Figure 1B ) in a patient with severe polycystic disease. Figure 1C : A dose-dependent mouse model of PC1 at 3 weeks of age. In the absence of Sec63, PC1 fails to mature efficiently in the ER, but in this case, PC1 matures efficiently by accumulating 3 additional genomic copies of Pkd1 (Pkd1 F / H -BAC) to produce more PC1 can prevent cyst formation. Ksp-Cre is active in the distal nephron starting in mid-embryonic period.

[0059] Figure 2A-2BCertain aspects of the 5'UTR and uORF of human PKD1 according to some embodiments are shown. Figure 2A : Linear sequence of the PKD1 mRNA start sequence showing the location of the uORF in the 5'UTR. Figure 2B : Predicted secondary structure of PKD1 5'UTR. Figure 2A Nucleotides 1-300 of SEQ ID NO: 1 are shown, which include the coding sequence of the first 30 amino acid residues of human PC-1 protein (MPPAAPARLALALGLGLWLGALAGGPGRGC, SEQ ID NO: 11). Figure 2B Nucleotides 1-209 of SEQ ID NO: 1 are shown.

[0060] Figure 3A-3B Shown are how uORFs function according to some embodiments and how antisense oligonucleotides (ASOs) inhibit certain aspects of uORFs. Figure 3A : Illustration of the deleterious effects of uORFs on protein translation. Figure 3B :ASO blocks uORF translation to increase protein translation.

[0061] Figures 4A-4D It is shown that, according to some embodiments, the uORF in the 5'UTR of human PKD1 downregulates translation and disruption of the uORF abrogates this downregulation. Figure 4A : Dual-luciferase reporter gene construction, in which the 5'UTR of Renilla luciferase was replaced by the wild-type 5'UTR or the 5'UTR mutant of human PKD1 with specific mutated uORFs. Figure 4B : Sequences of wild-type uORF ("wt") and mutant uORFs containing one ("ΔuORF1" and "ΔuORF2") or two ("ΔuORF1&2") single base edits to abolish one or both uORFs. Figure 4B In the figure, ΔuORF1 indicates an A to T mutation in nucleotide residue 123 of SEQ ID NO:2, ΔuORF2 indicates an A to T mutation in nucleotide residue 190 of SEQ ID NO:2, and ΔuORF1&2 indicates that both A to T mutations are included. Figure 4C : Targeted editing of the PKD1 5'UTR resulted in a significant increase in luciferase protein expression. Notably, abrogation of both uORFs increased protein expression by nearly 4-fold ("1&2" vs "wt"). Figure 4D : Mutation of uORF does not change mRNA expression.

[0062] Figure 5It was shown that, according to some embodiments, antisense oligonucleotides (ASOs) that inhibit uORF within the 5'UTR of PKD1 increase PC1 protein expression. Cells transfected with a wt-uORF luciferase reporter gene ( Figures 4A-4D ), nucleotide ASOs complementary to the mRNA sequence upstream of the uORF start codon of uORF1, uORF2, or both and overlapping the ATG of the start codon or mismatch control (MM1, MM2) ASOs were incubated with transfected cells. Lysates collected 24 hours after treatment showed a significant increase in luciferase expression (approximately 3-fold) when both uORF1 and 2 were targeted.

[0063] Figures 6A-6G It was shown that, according to some embodiments, ASO treatment in culture medium of human epithelial cells significantly increased PC1 protein expression. Fig. 6A :ASO1, ASO2 and their combination increased steady-state expression of PC1 without affecting mRNA expression. The protein expression data in this figure are from the 48-hour time point and are also included in Fig.6D middle. Figure 6B-6D : The increase in PC1 protein expression obtained after treatment with ASO1, ASO2 or their combination was most pronounced when the treatment was maintained for a longer period of time (expression levels normalized to the loading control Vinculin on western blots showed the strongest effects at 48 and 96 hours). Of note, fresh medium containing the specified concentrations of ASO was used once a day. Figure 6E-6G Different doses of ASO1 and ASO2 produced increased expression in cultured epithelial cells.

[0064] Figure 7 It was shown that, according to some embodiments, mouse polycystic kidney disease caused by epithelial cell-specific loss of the human polycystic kidney disease gene DNAJB11 was sensitive to a 50% reduction in the dose of PC1. This genetic interaction supports the speculation that a 2-fold increase in PC1 expression may be sufficient not only to slow cyst formation, but also to completely prevent cyst formation, as shown by preliminary in vitro data.

[0065] Figure 8 uORF1 and uORF2 in the 5'UTR of mouse Pkd1 mRNA according to some embodiments are shown. The mouse Pkd1 5'UTR contains two potential uORFs initiated by ATG, corresponding to the uORFs found in the human sequence. These two uORFs are named mouse uORF1 and mouse uORF2, which are consistent with the above-mentioned human uORFs. Figure 8 Residues 1-360 of SEQ ID NO:7 are shown, which include the coding sequence for the first 16 amino acid residues of the mouse PC1 protein (MPLGAPALLALALGLG, SEQ ID NO: 12).

[0066] Fig. 9 Shown is an ASO design for sterically blocking translation of the mouse Pkd1 uORF according to some embodiments. Fig. 9 Nucleotide residues 1-316 of SEQ ID NO: 7 are shown.

[0067] Figures 10A-10B According to some embodiments, PC1 protein expression after treatment with ASOs targeting mouse Pkd1 uORF in a mouse cell line containing an HA epitope tag at the C-terminus of Pkd1 to allow evaluation of the C-terminal fragment. All ASOs were at a concentration of 20 nM and were administered ( Fig. 10A )24 hours or ( Fig. 10B ) 96 hours for evaluation.

[0068] Fig.11 : According to some embodiments, PC1 protein expression after treatment with ASOs targeting the mouse Pkd1 uORF in a mouse cell line containing a missense mutation p.R2216W (homolog of the human variant p.R2220W) on the C-terminus of Pkd1 and a V5 epitope tag to allow evaluation of the C-terminal fragment. All ASOs were at a concentration of 20 nM; evaluation was performed 96 h after administration. These data indicate that inhibition of the Pkd1 uORF significantly increased the production of mature protein (PC1 C-terminal fragment), even in the presence of pathogenic missense variants.

[0069] Fig.12 The locations of three translation start sites that can be seen in public ribosome profiling experiments are shown, as visualized by the public Ribo-uORF resource. The uORF with evidence of translation in most experiments is our uORF1. Two additional uORFs (uORF3 and uORF4) with CTG start codons in the human PKD1 mRNA 5'-UTR were also identified from at least one ribosome profiling dataset. According to some embodiments, these can be targeted by ASOs to increase PC1 protein expression. Fig.12 The sequence shown is nucleotide residues 1-306 of SEQ ID NO: 1 (DNA nucleotide T replaces RNA nucleotide U). The numbering of uORFs has been edited according to the default numbering of the website, which is consistent with our numbering (reference: Liu et al., Nucleic Acids Research 2023). DETAILED DESCRIPTION

[0070] The following disclosure provides many different embodiments or examples for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. Of course, these are merely examples and are not intended to be limiting. For example, in the following description, forming a first feature on or on a first feature may include an embodiment in which the first and second features are directly contacted to form, and may also include an embodiment in which additional features are formed between the first and second features. In addition, the present disclosure may repeat reference numbers and / or letters in various examples. This repetition is for simplicity and clarity, and does not itself determine the relationship between the various embodiments and / or configurations discussed.

[0071] PCLD, as well as an important subset of ADPKD, is caused by insufficient dosage of PC1 function, either due to loss-of-function mutations in the PKD1 gene itself or mutations in endoplasmic reticulum (ER) genes responsible for sorting PKD1 to the cell surface of primary cilia.

[0072] It is speculated that both PCLD and ADPKD caused by insufficient PC1 functional dosage can be treated, improved and / or prevented by increasing the patient's PC1 functional dosage. The only known way to increase PC1 expression is to inhibit microRNA17 (Lakhia et al., "PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression"). The proposed therapy is also at the level of preclinical research in mouse models and is based on blocking the steady-state inhibition of PKD1 mRNA by microRNA17 binding to the 3'-UTR of the PKD1 transcript. In vivo studies have shown promise for methods to therapeutically increase PC1 dosage. Since complementary sequences to the microRNA17 sequence are present in a large number of genes, the use of this molecule to treat PCLD and ADPKD is expected to have significant and unpredictable off-target effects, i.e., lack of specificity.

[0073] In this study, four upstream open reading frames (uORFs) were found in the 5'-untranslated region (5'-UTR) of PKD1 messenger RNA (mRNA). This study further found that the translation of uORF1 and / or uORF2 resulted in reduced translation of PKD1 mRNA into PC1 protein, and that abrogation of these two uORFs by mutation or blocking their translation using antisense oligonucleotides (ASOs) significantly enhanced the translation of PKD1 mRNA, thereby increasing PC1 protein levels. It is expected that the functions of the third and fourth uORFs may be similar or complementary to those of the first and second uORFs, and that abrogation and / or inhibition of the third and fourth uORFs may achieve similar or enhanced effects. Since the uORFs on the 5'-UTR of PKD1 have unique sequences, and methods targeting the uORF sequences, such as CRISPR or ASO, can be designed to be highly specific, the treatment of ADPKD or PCLD by uORFs is expected to be highly specific.

[0074] Therefore, in some aspects, the present invention relates to methods for treating, ameliorating and / or preventing polycystic kidney disease or polycystic liver disease.

[0075] In some aspects, the invention relates to methods of increasing the functional dose of PC1 in a cell, tissue or subject.

[0076] definition

[0077] As used herein, each of the following terms has the meaning associated therewith in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In general, the terms used herein and laboratory procedures in animal pharmacology, pharmaceutical sciences, peptide chemistry, and organic chemistry are well known and commonly used in the art. It should be understood that the order of steps or the order in which certain actions are performed is unimportant as long as the present teachings remain feasible. The use of section headings is to assist in reading the document and should not be construed as limiting; information associated with section headings may appear within or outside that particular section. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if incorporated herein by reference alone.

[0078] In the present application, when an element or component is considered to be included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component can be any one of the enumerated elements or components and can be selected from a group consisting of two or more enumerated elements or components.

[0079] In the methods described herein, actions may be performed in any order unless a time or sequence of operations is explicitly referenced. In addition, specific actions may be performed simultaneously unless explicit claim language details that they may be performed separately. For example, a claimed action of doing X and a claimed action of doing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed process.

[0080] In this document, the terms "a", "an" or "the" are used to include one or more, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise stated. The expression "at least one of A and B" or "atleast one of A or B" has the same meaning as "A, B, or A and B".

[0081] As used herein, "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to include variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, such that these variations are suitable for performing the disclosed methods.

[0082] As used herein, "PKD1" refers to the human gene that is transcribed to produce the mRNA product represented by SEQ ID NO: 1, all human genes that have the same alleles as the human gene that produces the mRNA of SEQ ID NO: 1; all orthologous genes in non-human species, and all mRNA products and protein products of human and non-human genes (sometimes referred to in the art and herein as "polycystin-1", "polycystin 1", "PC-1" or "PC1" protein).

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] In some embodiments, PKD1 refers to human PKD1 having a 5'UTR, and its RNA sequence is shown in SEQ ID NO:2.

[0093]

[0094] In some embodiments, PKD1 refers to human PKD1 having the following upstream open reading frame in its 5'UTR:

[0095]

[0096] In some embodiments, Pkd1 refers to mouse Pkd1 having the following mRNA sequence:

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] In some embodiments, Pkd1 refers to mouse Pkd1 having a 5'UTR having the RNA sequence set forth in SEQ ID NO:8.

[0107]

[0108] In some embodiments, Pkd1 refers to mouse Pkd1 having the following upstream open reading frame in its 5'UTR:

[0109]

[0110] For the purpose of this specification, the term "first uORF", "second uORF", "third uORF" or "fourth uORF" does not mean that these uORFs are located in this order in the mRNA sequence. Instead, the term "first uORF", "second uORF", "third uORF" and "fourth uORF" refer to uORFs having the above sequence in the 5'UTR of PKD1 mRNA, or uORFs at the same or similar position in the 5'UTR of PKD1 mRNA and having a homologous sequence (such as having a sequence similarity of about 80% or more, 85% or more, 90% or more or 95% or more).

[0111] 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.

[0112] Methods for treating, ameliorating and / or preventing ADPKD or PCLD

[0113] In some aspects, the present invention relates to a method of treating, ameliorating and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof.

[0114] In some embodiments, the method comprises administering to the subject an effective amount of a compound that inhibits translation of or affects secondary mRNA structure of the first upstream open reading frame (uORF), the second uORF, the third uORF, or the fourth uORF, or a combination thereof, of the PKD1 gene.

[0115] In some embodiments, the method is a method of treating, ameliorating and / or preventing ADPKD in a subject, and the ADPKD is caused by or involves a mutation in a PKD1 or PKD2 gene of the subject.

[0116] In some embodiments, the method is a method for treating, ameliorating and / or preventing PCLD in a subject, and wherein PCLD is caused by or involves 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 (including those genes that have not yet been determined for the subject's ADPKD-PCLD disease spectrum).

[0117] In some embodiments, the compound that inhibits the first upstream open reading frame (uORF), the second uORF, the third uORF and / or the fourth uORF of the PKD1 gene comprises:

[0118] 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 that expresses the CRISPR component; or

[0119] an antisense oligonucleotide (ASO) that blocks the first uORF, the second uORF, the third uORF and / or the fourth uORF,

[0120] Or an expression vector expressing the ASO.

[0121] This study determined the sequences of four uORFs in the 5'-UTR of PKD1 mRNA and demonstrated that inhibition of either or both of the first two uORFs by altering the sequence of the uORFs or blocking the uORFs with ASOs significantly increased PC1 expression. It is expected that the third and fourth uORFs in PKD1 mRNA may function similarly or complementary to the first and second uORFs, and that abrogation or inhibition of uORFs 3 and 4 will achieve similar or enhanced effects.

[0122] A person of ordinary skill in the art will understand that the techniques for designing CRISPR systems and ASOs are known in the art, and that, through the target sequences of the uORFs determined in this study and the description of CRISPR and ASOs herein (e.g., the sections “Inhibiting PKD1 uORF by CRISPR technology” and “Inhibiting PKD1 uORF by ASO technology”), CRISPR components and ASOs can be designed and obtained without performing unnecessary experiments.

[0123] In some embodiments, the compound includes a CRISPR component or an expression vector expressing 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.

[0124] In some embodiments, the compound includes an ASO or an expression vector expressing an ASO, and the portion of the PKD1 mRNA complementary to the ASO extends from the start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF to 5 nucleotides or less, such as to 4 nucleotides or less, to 3 nucleotides or less, to 2 nucleotides or less, to 1 nucleotide or less, to the boundary of the start codon, to 1 nucleotide or more of the start codon, to 2 nucleotides or more of the start codon, or to the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

[0125] In some embodiments, the ASO inhibits the first uORF ("uORF 1"). In some embodiments, the ASO targets a sequence (SEQ ID NO: 13, the start codon is highlighted), which includes the 5'-portion of uORF 1, as well as some nucleotides to its 5'-end.

[0126] In some embodiments, the 5'-UTR sequence that is complementary to (e.g., about 80% or more complementary, about 85% or more complementary, about 90% or more complementary, about 95% or more complementary, or completely complementary to) the ASO and targeted by the ASO is at least one of the following:

[0127]

[0128]

[0129] In some embodiments, the ASO inhibits uORF 2. In some embodiments, the ASO targets a sequence (SEQ ID NO:, the start codon is highlighted), which includes the 5'-portion of uORF 2, and some nucleotides to its 5'-end.

[0130] In some embodiments, the sequence of the 5'-UTR that is complementary to the ASO (e.g., 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:

[0131]

[0132]

[0133]

[0134] In some embodiments, the ASO inhibits uORF 3. In some embodiments, the ASO targets a sequence (SEQ ID NO:, the start codon is highlighted), which includes the 5'-portion of uORF 3, and some nucleotides to its 5'-end.

[0135] In some embodiments, the sequence of the 5'-UTR that is complementary to the ASO (e.g., 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:

[0136]

[0137]

[0138]

[0139] In some embodiments, in an ASO that is "fully complementary to" one of the sequences listed in SEQ ID NOs: 14-157, every base participates in hybridization with a base in that sequence, and vice versa.

[0140] In some embodiments, the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer, or 15 nucleotides or longer.

[0141] In some embodiments, the ASO is 30 nucleotides or less in length, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0142] In some embodiments, the ASO is an RNA molecule, a DNA molecule, or a DNA-RNA hybrid molecule. In some embodiments, the ASO includes a modified base, a modified linkage, and / or a modified sugar group.

[0143] In some embodiments, the modified sugar group includes a 2'-O-methylated (2'-OMe) modified sugar group, a locked nucleic acid (LNA) modified sugar group, a 2'-O-methoxyethyl (2'-MOE) modified sugar group, a (S)-constrained ethyl nucleic acid (cEt) modified sugar group, or a 2' fluorine (2'F) modified sugar group.

[0144] In some embodiments, the modified linkage includes phosphorothioate (PS) linkage, phosphorodiamidate morpholino (PMO) linkage, positively charged PMO linkage, phosphoramide linkage, methylphosphonate (MP) linkage, phosphorothioate linkage or peptide nucleic acid (PNA) linkage. In some embodiments, the ASO includes a combination of modified linkage and natural phosphodiester (PO) linkage, such as a combination of PS linkage and PO linkage. In some embodiments, the change can cause it to be characterized as a PMO morpholino.

[0145] In some embodiments, the modified bases include 5' methylcytosine bases, or G-clamp bases.

[0146] In some embodiments, the ASO is conjugated to an N-acetylgalactosamine (GalNAc) group, or a cholesterol group. In some embodiments, conjugation improves the delivery of the ASO, such as delivery across a cell membrane.

[0147] In some embodiments, the ASO is designed using a Gapmer. In some embodiments, the Gapmer includes an internal "gap" based on DNA and an RNA-like flanking region. In some embodiments, the RNA-like flanking region includes one or more modifications, such as the modifications described above, such as 2'-OMe or LNA modifications.

[0148] In some embodiments, the ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159), or a variant thereof. It is noteworthy that since ASOs can be RNA-based, DNA-based, or DNA / RNA hybrid-based, and can include modifications of bases, linkages, or sugar groups, the ASO molecules herein are not intended to be limited to the molecular properties indicated by the listed sequences. For example, although the sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158) indicates that the present ASO molecule is an RNA molecule, the present sequence is intended to also include the corresponding DNA molecules, the corresponding DNA / RNA hybrid molecules, and the corresponding nucleic acids including modified bases, linkages, and / or sugar groups.

[0149] In some embodiments, the ASO concentration in the affected tissue (kidney or lung) after administration ranges from 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 nm to about 20 nm.

[0150] In some embodiments, the subject is a mammal, such as a human.

[0151] Methods for increasing the expression of PKD1 / polycystin-1 (PC1) in cells

[0152] In some embodiments, the disclosure relates to a method of increasing PKD1 / PC1 expression in a cell.

[0153] In some embodiments, the method comprises: contacting a cell with an effective amount of a compound that inhibits a first upstream open reading frame (uORF) and / or a second uORF of a PKD1 gene.

[0154] 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.

[0155] In some embodiments, the cell is located in a tissue or a subject.

[0156] In some embodiments, the cell is a kidney cell from a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or a liver cell from a subject diagnosed with polycystic liver disease (PCLD).

[0157] In some embodiments, the compound comprises:

[0158] A CRISPR component or an expression vector expressing the CRISPR component, the CRISPR component disrupting the genomic DNA sequence encoding the first uORF and / or the second uORF; or

[0159] An antisense oligonucleotide (ASO) or an expression vector expressing the ASO, wherein the ASO blocks the first uORF and / or the second uORF.

[0160] Such CRISPR components and ASOs are described in detail elsewhere herein.

[0161] In some embodiments, the compound includes a CRISPR component or an expression vector expressing a CRISPR component, and the CRISPR component disrupts the start codon of the first uORF and / or the second uORF.

[0162] In some embodiments, the compound includes an ASO or an expression vector expressing an ASO, and the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less from the start codon of the first uORF or the second uORF, such as extending to 4 nucleotides or less, extending to 3 nucleotides or less, extending to 2 nucleotides or less, extending to 1 nucleotide or less, reaching the boundary of the start codon, reaching 1 nucleotide or more of the start codon, reaching 2 nucleotides or more of the start codon, or reaching the entire start codon of the first uORF or the second uORF.

[0163] In some embodiments, the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer, or 15 nucleotides or longer.

[0164] In some embodiments, the ASO is 30 nucleotides or less in length, such as 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, or 25 nucleotides or less.

[0165] In some embodiments, the ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159), or variants thereof, including modified bases or modified linkages.

[0166] In some embodiments, the modified bases include 2'-O-methylated modified bases.

[0167] In some embodiments, the modified linkage comprises a phosphorothioate (PS) linkage.

[0168] In some embodiments, the concentration of the ASO contacted with the cell ranges from 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 nm to about 20 nm.

[0169] Inhibition of PKD1 uORF by CRISPR technology

[0170] In some embodiments, the CRISPR components are designed to induce targeted genetic alterations of a uORF sequence in the 5'-UTR of the PKD1 gene, such as the start codon ("AUG / ATG") of the uORF in genomic DNA.

[0171] The CRISPR / Cas9 system is a simple and efficient system for inducing targeted genetic changes. Target recognition by the Cas9 protein requires a "seed" sequence in the guide RNA (gRNA) and a conserved dinucleotide sequence containing a protospacer adjacent motif (PAM) upstream of the gRNA binding region. Thus, by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells, the CRISPR / Cas9 system can be designed to cut essentially any DNA sequence. The CRISPR / Cas9 system can simultaneously target multiple genomic sites by co-expressing a single Cas9 protein and two or more gRNAs, making the system uniquely suitable for multi-gene editing or coordinated activation of target genes.

[0172] Cas9 protein and guide RNA form a complex that recognizes and cuts the target sequence. Cas9 consists of six domains: REC I, REC II, 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 and RuvC domains are nuclease domains. The guide RNA is designed so that its 5' end is complementary to the target DNA sequence. After the guide RNA binds to the Cas9 protein, a conformational change occurs, thereby activating the protein. Once activated, Cas9 binds to a sequence that conforms to its original spacer adjacent motif (PAM) sequence to search for target DNA. PAM is a two or three nucleotide base sequence within one nucleotide downstream of the complementary region of the guide RNA. In a non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds a target sequence with an appropriate PAM, it melts the base upstream of the PAM and pairs it with the complementary region on the guide RNA. The RuvC and HNH nuclease domains then cleave the target DNA after the third nucleotide base upstream of the PAM.

[0173] A non-limiting example of a CRISPR / Cas system (CRISPRi) for inhibiting gene expression is described in U.S. Patent Application Publication No. US2014 / 0068797. CRISPRi uses RNA-guided Cas9 endonucleases to induce permanent gene damage, introducing DNA double-strand breaks, which trigger error-prone repair pathways to cause frameshift mutations. Catalytically dead Cas9 lacks endonuclease activity. When co-expressed with guide RNA, a DNA recognition complex is generated that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system effectively inhibits the expression of target genes.

[0174] When the guide nucleic acid sequence and Cas endonuclease specific to the target gene are introduced into the cell and form a complex, so that the Cas endonuclease can introduce a double-strand break on the target gene, CRISPR / Cas gene disruption occurs. 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 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 ribozymes known in the art, and any combination thereof.

[0175] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to a preparation that activates an inducible promoter in the Cas expression vector. In this embodiment, the Cas expression vector comprises an inducible promoter, such as a promoter that can be induced by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be understood that other inducible promoters can be used. The inducer can be a selective condition that causes the inducible promoter to be induced (e.g., exposure to a preparation such as an antibiotic). This results in expression of the Cas expression vector.

[0176] In certain embodiments, guide RNA(s) and Cas9 can be delivered to cells as a ribonucleoprotein (RNP) complex. RNP consists of a complex of purified Cas9 protein and gRNA, and is well known in the art to be effectively delivered to various types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI).

[0177] The guide RNA is specific to the target genomic region and performs Cas endonuclease-induced double-strand breaks on the region. The target sequence of the guide RNA sequence can be located within a gene locus or within a non-coding region of the genome. In certain embodiments, the length of 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.

[0178] Guide RNA (gRNA), also known as "short guide RNA" or "sgRNA", provides targeting specificity and scaffolding / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA consisting of a target sequence and a scaffolding sequence, and the scaffolding sequence is derived from endogenous bacterial crRNA and tracrRNA. The gRNA is used to target Cas9 to a specific genomic site in genome engineering experiments. Guide RNAs can be designed using standard tools known in the art.

[0179] In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, wherein hybridization between the target sequence and the guide sequence can promote the formation of a CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may include any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm. In other embodiments, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or a nucleus. Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in the cleavage of one or both chains 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). As with the target sequence, it is believed that complete complementarity is not required, as long as the complementarity is sufficient to function.

[0180] In certain embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell so that the expression of the CRISPR system elements guides the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence connected to the tracr-mate sequence, and the tracr sequence can be connected to different regulatory elements on different vectors, respectively. Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, wherein one or more additional vectors provide any element 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 direction, such as one element is located 5' ("upstream") or 3' ("downstream") of the second element. The coding sequence of an element can be located on the same or opposite chain of the coding sequence of the second element, and in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more guide sequences, a tracr partner sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intronic sequences (e.g., each in a different intron, two or more in at least one intron, or all in one intron).

[0181] In certain embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may include any additional protein sequences and may optionally also include a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, 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. Other domains that may constitute a part of a fusion protein including a CRISPR enzyme are described in U.S. Patent Application Publication No. US20110059502, which is incorporated herein by reference. In certain embodiments, a labeled CRISPR enzyme is used to identify the location of a target sequence.

[0182] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. These methods can be used to introduce nucleic acids encoding CRISPR system components into cultured cells or host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (transcripts of vectors described herein), naked nucleic acids, and nucleic acids compounded with delivery vectors (such as liposomes). Viral vector delivery systems include DNA and RNA viruses, which have exogenous or integrated genomes after delivery to cells (Anderson, 1992, Science 256: 808-813; Yu et al., 1994, Gene Therapy 1: 13-26).

[0183] In some embodiments, CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0184] In general, the Cas protein includes at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guide RNA. The Cas protein may also include a nuclease domain (i.e., a 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. The Cas protein may be modified to increase nucleic acid binding affinity and / or specificity, change enzyme activity, and / or change another property of the protein. In certain embodiments, the Cas protein of the fusion protein may be derived from a wild-type Cas9 protein or a fragment thereof. In other embodiments, Cas may be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein may be modified to change one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, the domains of the Cas9 protein that are not involved in RNA-guided cleavage may be eliminated, so that the modified Cas9 protein is smaller than the wild-type Cas9 protein. In general, the Cas9 protein includes at least two nuclease (i.e., DNase) domains. For example, the Cas9 protein may include a RuvC-like nuclease domain and a HNH-like nuclease domain. RuvC and HNH domains jointly cut single strands to break DNA double strands. (Jinek et al., 2012, Science, 337: 816-821). In some embodiments, the Cas9 derivative protein may be modified to contain only one functional nuclease domain (RuvC-like or HNH-like nuclease domain). For example, the Cas9 derivative protein may be modified to delete or mutate one of the nuclease domains so that it is no longer functional (i.e., it has no nuclease activity). In some embodiments in which one of the nuclease domains is inactive, the Cas9 derivative protein can introduce a gap into a double-stranded nucleic acid (this protein is called a "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 full gene synthesis, as well as other methods known in the art, may be used to inactivate any or all of the nuclease domains by one or more deletion mutations, insertion mutations and / or substitution mutations.

[0185] In a non-limiting embodiment, the vector drives the expression of the CRISPR system. There are a large number of vectors suitable for this specification in the art. The vector to be used is suitable for replication and, optionally, for integration into eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence. The vectors in this specification can also be used for nucleic acid standard gene delivery protocols. Gene delivery methods are known in the art (U.S. Patents 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference).

[0186] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, such as described in Sambrook et al. (4th edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus, Sindbis virus, gamma retrovirus and slow virus. In general, suitable vectors include replication origins, promoter sequences, 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) that work in at least one organism.

[0187] In some embodiments, the present invention includes any other method for achieving gene knockout and / or editing that can achieve deletion and / or inactivation of uORFs, such as but not limited to the method described in WO 2018 / 236840 (the entire contents of which are incorporated herein by reference).

[0188] Inhibition of PKD1 uORF by ASO technology

[0189] In some embodiments, the ASO molecule is designed so that the ASO hybridizes to the 5'-UTR of PKD1 mRNA at or near the uORF (eg, at or near the start codon of the uORF), thereby greatly reducing the ribosome binding efficiency of the uORF.

[0190] Antisense oligonucleotides (ASOs) are short chains of nucleotide analogs that hybridize to complementary mRNA in a sequence-specific manner. The technology was first described in the 1970s and has been developing since the 1980s with advances in oligonucleotide chemistry and formulation technology. Currently, several ASO drugs have been approved and a large number of ASO drugs are under development. (Bennett, Annual Review of Medicine, Vol. 70: 307-321, 2019).

[0191] The length of ASO is usually 13-25 nucleotides, but is not limited thereto. ASOs are complementary to the mRNA they target and can hybridize with the mRNA through Watson-Crick base pairing. The sequence of ASOs can be designed with the aid of existing software and algorithms. For example, Chan et al. (CEPP, Vol. 33, May 5-June 5, 2006, pp. 533-540) describe the design of ASOs for specific sequences.

[0192] The formation of mRNA / ASO pairs can produce two effects: 1) ribonuclease H (RNaseH)-mediated cleavage, which degrades the pair; and 2) steric hindrance, which restricts ribosome access to the mRNA segment complementary to the ASO.

[0193] Because, according to certain embodiments, it is desirable to provide steric hindrance to limit access to the uORF in the 5'-UTR of the PKD1 mRNA (which will increase translation of PC1 protein from the PKD1 mRNA) without causing degradation of the mRNA (which will reduce expression of the PKD1 mRNA and its translation into PC1 protein), in certain embodiments, the ASO herein includes a chemical modification that renders the formed RNA / ASO pair resistant to endonuclease cleavage. Sugar modifications, such as 2'-O-methyl (2'-OMe) or 2'-O-methoxyethyl (2'-O-MOE) modifications and phosphorothioate modifications, as well as linkage modifications, such as phosphorothioate (PS) modifications, are known to increase resistance of RNA / ASO pairs to endonuclease cleavage (see, e.g., Gagliardi et al., Biomedicines. 2021 Apr; 9(4): 433 and Crooke et al., Nucleic Acids Res. 2022 Jun 4: 5235-5253). Thus, in some embodiments, the ASOs herein include one or more of such modifications.

[0194] Roberts et al. (Nature Reviews Drug Discovery, Vol. 19, pp. 673-694 (2020)) also described ASO design for improved delivery.

[0195] Carrier

[0196] A vector may increase the stability of a nucleic acid, make delivery easier, or allow expression of the nucleic acid or its protein product in a cell.

[0197] Thus, in some embodiments, a compound that inhibits the PKD1 uORF is incorporated into a vector.

[0198] In some embodiments, this specification relates to vectors, including the nucleic acid sequences of this specification or the construction of this specification. The selection of vectors depends on the host cell to be subsequently introduced. In certain embodiments, the vector of this specification is an expression vector. Suitable host cells include various 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. Systems based on prokaryotic and / or eukaryotic vectors can be used for the production of polynucleotides or their homologous polypeptides in this specification. Many such systems can be widely available on the market.

[0199] In certain embodiments, the vector is a viral vector. Viral vector technology is well known in the art, such as described in the Handbook of Virology and Molecular Biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers that function in at least one organism. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0200] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the adeno-associated virus AAV1-AAV8 family. In some embodiments, the viral vector is a viral vector that can infect humans. The desired nucleic acid sequence (such as the above 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 a thyroxine binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desired nucleic acid to be expressed in the liver. AAV can be a recombinant AAV, in which the capsid is from one AAV serotype and the ITR is 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 ITR in AAV is at least one ITR selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 ITRs. 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, when administered to a subject, can cause elevated systemic expression of the desired nucleic acid. 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.

[0201] In some embodiments, the vector for introducing the nucleic acid sequence is a plasmid, which may or may not be integrated into the genome of the host cell when introduced into the host cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of this specification or the gene construction of this specification can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0202] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0203] In certain embodiments, the recombinant expression vector may further comprise a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor of the present specification, which is described elsewhere herein.

[0204] A promoter may be one naturally associated with a gene or polynucleotide sequence, which may be obtained by isolating a 5' non-coding sequence upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide fragment under the control of a recombinant or heterologous promoter, which refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters and enhancers isolated from any other prokaryotic, viral or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., different elements containing different transcriptional regulatory regions, and / or mutations that alter expression. In addition to synthesizing nucleic acid sequences that produce promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR) may also be used. TM ) in combination with the compositions disclosed herein to produce sequences (U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906). In addition, control sequences that direct transcription and / or expression of sequences in non-nuclear organelles such as mitochondria and chloroplasts can also be considered.

[0205] It is important to use promoters and / or enhancers that effectively direct the expression of the DNA fragment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations to express proteins. The promoter used can be constitutive, tissue-specific, inducible, and / or can be used to direct high-level expression of the introduced DNA fragment under appropriate conditions, such as having advantages in large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0206] The recombinant expression vector may also contain a selectable marker gene, which is convenient for selecting a host cell for transformation or transfection. Suitable selectable marker genes are genes encoding proteins, such as G418 and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or immunoglobulin or its portion, such as the Fc portion (preferably IgG) of an immunoglobulin, which are resistant to certain drugs. Selectable markers may be introduced into a separate vector together with the target nucleic acid.

[0207] Combination therapy

[0208] In some embodiments, a method of treating, ameliorating 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.

[0209] In some embodiments, a composition for treating polycystic disease includes at least one compound and / or composition contemplated within the present disclosure.

[0210] In some embodiments, the subject is further administered at least one additional agent for treating, ameliorating and / or preventing the diseases and / or disorders contemplated herein. 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.

[0211] The compounds contemplated in the present disclosure are intended to be used in combination with one or more additional compounds. These additional compounds may include compounds of the present disclosure 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.

[0212] The synergy effect can be calculated using suitable methods, for example, Sigmoid-E max Equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6: 429-453), Loewe addition equation (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and median effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each of the above equations can be applied to experimental data to generate corresponding charts to help evaluate the effect of drug combinations. The corresponding charts associated with the above equations are concentration-effect curves, isohologram curves, and combination index curves.

[0213] Administration / dosage / formulation

[0214] The administration regimen may affect what constitutes an effective amount. The therapeutic formulations contemplated by the present disclosure may be administered to a subject before or after the onset of a disease and / or disorder. In addition, several divided doses and staggered doses may be administered daily or sequentially, or the dose may be infused continuously or may be a bolus injection. In addition, the dosage of the therapeutic formulations contemplated in the present disclosure may be proportionally increased or decreased as required by the treatment or prevention situation.

[0215] The compositions contemplated in the present disclosure can be administered to patients (preferably mammals, more preferably humans) using known procedures, dosages, and time periods to effectively treat the diseases and / or disorders contemplated in the patients. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary according to factors such as the disease or disorder state of the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound described in the present disclosure to treat the patient's disease and / or disorder. The dosage regimen can be adjusted to provide the best therapeutic response. For example, the dosage can be divided into several doses per day, or the dosage can be reduced proportionally as required by the treatment situation. A non-limiting example of an effective dosage range for the therapeutic compound contemplated in the present disclosure is about 1 to 5000 mg / kg body weight / day. A person of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the therapeutic compound without unnecessary experimentation.

[0216] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective for a particular patient, composition, and mode of administration to achieve the desired therapeutic response without causing toxicity to the patient.

[0217] In particular, the selected dosage level will depend upon a variety of factors, including the activity of the specific compound employed, the time of administration, the excretion rate of the compound, the duration of the treatment, other drugs, compounds or materials used in conjunction with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0218] A physician having ordinary skill in the art, such as a physician or veterinarian, can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the compound contemplated by the present disclosure employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0219] 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, dosage unit form refers to physically discrete units suitable as unit dosages for 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 carrier. The dosage unit forms contemplated in this disclosure are determined by and directly dependent on (a) the unique characteristics of the therapeutic compound and the specific 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.

[0220] 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 include a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0221] The carrier can be a solvent or dispersion medium, for example containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained by using coatings such as lecithin, by maintaining the required particle size and using surfactants in the case of dispersion. The effect of microorganisms can be prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents in the composition, such as sugar, sodium chloride or polyols such as mannitol and sorbitol. The absorption time of the injectable composition can be extended by including a medicament (such as aluminum monostearate or gelatin) that delays absorption in the composition.

[0222] In certain embodiments, the compositions of the present disclosure are administered to the patient at a dosage of 1 to 5 times or more per day. In another embodiment, the compositions of the present disclosure are administered to the patient at a dosage range including, but not limited to, once a day, once every two days, every three days to once a week, once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various compositions of the present disclosure varies from person to person, depending on many factors, including, but not limited to, age, disease or disorder to be treated, sex, general health, and other factors. Therefore, the present disclosure should not be construed as being limited to any particular dosage regimen, and the precise dosage and composition administered to any patient is determined by the attending physician after considering all other factors concerning the patient.

[0223] The compounds of the present disclosure for administration may range from about 1 μg to about 10000 mg, about 20 μg to about 9500 mg, about 40 μg to about 9000 mg, about 75 μg to about 8500 mg, about 150 μg to about 7500 mg, about 200 μg to about 7000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5000 mg, about 750 μg to about 4000 mg, about 1 mg to about 3000 mg, about 10 mg to about 2500 mg, about 20 mg to about 2000 mg, about 25 mg to about 1500 mg, about 30 mg to about 1000 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 any and all whole or partial increments therebetween.

[0224] In some embodiments, the dosage of the disclosed compounds is from about 1 mg to about 2500 mg. In some embodiments, the dosage of the disclosed compounds used in the compositions described herein is less than about 10000 mg, or less than about 8000 mg, or less than about 6000 mg, or less than about 5000 mg, or less than about 3000 mg, or less than about 2000 mg, or less than about 1000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Likewise, in some embodiments, the dose of the second compound described herein is less than about 1000 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 or partial increments thereof.

[0225] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present disclosure, alone or in combination with a second agent; and instructions for using the compound to treat, prevent, or alleviate one or more symptoms of polycystic disease in a patient.

[0226] The preparation can be mixed with conventional excipients (i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for intracranial, oral, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable administration known in the art). The pharmaceutical preparation can be sterilized and, if necessary, can also be mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, buffer salts that affect osmotic pressure, colorants, flavorings and / or aromatic substances, etc. When necessary, they can also be used in combination with other active agents (e.g., other analgesics).

[0227] The administration route of any composition of the present disclosure includes oral, nasal, rectal, vaginal, parenteral, buccal, sublingual or topical. The compound used in the present disclosure can be formulated into any suitable route for administration, such as oral or parenteral administration, such as transdermal, transmucosal (e.g., sublingual, lingual, (through) oral, (through) urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal (intra) and (through) rectal), intravesical, intrapulmonary, intraduodenal, intragastrointestinal, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation and topical administration.

[0228] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, tablets, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, pastes, lozenges, creams, ointments, patches, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0229] Oral administration

[0230] Particularly suitable for oral administration are tablets, troches, liquids, drops, suppositories or capsules, caplets and gel caps. Compositions for oral administration may be prepared according to any method known in the art, such compositions may contain one or more formulations selected from the group consisting of inert, non-toxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients include, for example, inert diluents, such as lactose; granulating and disintegrants, such as corn starch; binders, such as starch; and lubricants, such as magnesium stearate. Tablets may be uncoated or coated by known techniques to maintain elegance or to delay the release of the active ingredient. Oral preparations may also be hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0231] For oral administration, the compounds of the present disclosure can be prepared into tablets or capsules by conventional methods and added with pharmaceutically acceptable excipients, such as binders (e.g., polyvinyl pyrrolidone, hydroxypropyl cellulose or hydroxypropyl methylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, the tablets can be coated using suitable methods and coating materials, such as OPADRY available from Colorcon, West Point, Pennsylvania, USA TM Film coating systems (eg OPADRY TM OY type, OYC type, organic enteric-coated OY-P type, aqueous enteric-coated OY-A type, OY-PM type and OPADRY TM White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups or suspensions. Liquid preparations may be prepared by conventional methods and may contain pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters or ethanol); and preservatives (e.g., methyl or propyl paraben or sorbic acid).

[0232] The present disclosure also includes multilayer tablets comprising a layer for delayed release of one or more compounds of the present disclosure and a further layer for immediate release of another drug. The use of wax / pH sensitive polymer compositions allows for the achievement of gastro-insoluble compositions in which the active ingredient is embedded, ensuring its delayed release.

[0233] Parenteral administration

[0234] For parenteral administration, the compounds of the present disclosure can be formulated as injections or infusions, such as intravenous, intramuscular or subcutaneous injections or infusions, or administered in single doses and / or continuous infusions. Suspensions, solutions or emulsions in oily or aqueous vehicles can also be used, optionally containing other agents such as suspending agents, stabilizers and / or dispersants.

[0235] Other forms of administration

[0236] Other dosage forms disclosed herein 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. Other dosage forms disclosed herein also include those described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688 and 20020051820. Other dosage forms disclosed herein 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.

[0237] Controlled release formulations and drug delivery systems

[0238] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-release, and controlled, for example, sustained-release, delayed-release, and pulsatile-release formulations.

[0239] The term "sustained release" in the conventional sense refers to a pharmaceutical preparation that gradually releases a drug over a long period of time, and it can (but not necessarily) keep the blood concentration of the drug substantially constant over a long period of time. The release time can be as long as one month or longer, and the release time should be longer than the same amount of drug administered in the form of a suppository.

[0240] To achieve sustained release, the compound may be formulated with a suitable polymer or hydrophobic material to provide sustained release properties. Thus, the compound used in the disclosed method may be administered in microparticle form, for example by injection or by implantation in the form of a wafer or disk.

[0241] In certain embodiments of the present disclosure, a compound of the present disclosure, alone or in combination with another pharmaceutical agent, is administered to a patient using a sustained release formulation.

[0242] The term "delayed release" as used herein in its conventional sense refers to a drug formulation that provides initial release of the drug after a delay after drug administration, and which may (but does not necessarily) include a delay of about 10 minutes to about 12 hours.

[0243] As used herein, the term "pulsatile release" refers in a conventional sense to a drug formulation that releases a drug in a manner that produces a pulsatile plasma profile following drug administration.

[0244] The term "immediate release" refers to a drug formulation that releases the drug immediately after administration.

[0245] As used herein, short-term refers to any time period after drug administration up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any or all whole or partial increments thereof.

[0246] As used herein, rapid compensation refers to any time period after drug administration up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any or all full or partial increments thereof.

[0247] dose

[0248] The therapeutically effective amount or dosage of the compounds disclosed herein depends on the age, sex and weight of the patient, the patient's current physical condition and the progression of the patient's polycystic disease. A skilled technician can determine the appropriate dosage based on these and other factors.

[0249] The range of suitable dosages of the compounds of the present disclosure may be from about 0.01 mg to about 5000 mg per day, such as from about 0.1 mg to about 1000 mg per day, for example, from about 1 mg to about 500 mg per day, such as from about 5 mg to about 250 mg per day. The dosage may be a single administration or multiple administrations, for example, from 1 to 4 times per day or more. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dosage of 1 mg per day may be administered in two portions, 0.5 mg per administration, with an interval of about 12 hours between each administration.

[0250] It is understood that, in non-limiting examples, the amount of compound administered daily can be every day, every other day, every 2 days, every 3 days, every 4 days or every 5 days. For example, in the case of administration every other day, 5 mg can be administered daily starting on Monday, 5 mg can be administered for the first subsequent day on Wednesday, 5 mg can be administered for the second subsequent day on Friday, and so on.

[0251] If the patient's condition does improve, the doctor may optionally continue to administer the modulator of the present disclosure, or temporarily reduce the dosage or temporarily stop the medication for a period of time (i.e., a "rest period"). The length of the rest period optionally varies 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. Dose reductions during the rest period include from 10%-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%.

[0252] Once the patient's condition has improved, a maintenance dose may be administered if necessary. Subsequently, depending on the patient's condition, the dosage or frequency of administration may be reduced, or both, to maintain the level of disease improvement. In certain embodiments, the patient requires long-term intermittent treatment upon recurrence of any symptoms and / or infection.

[0253] The compounds used in the disclosed methods can be formulated into unit dosage forms. The term "unit dosage form" refers to physically discrete units suitable for use as unit doses for patients receiving treatment, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, optionally in combination with an appropriate pharmaceutical carrier. The unit dosage form can be used for a single daily dose or multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple doses are used per day, the unit dosage form of each dose can be the same or different.

[0254] Optionally, the toxicity and therapeutic efficacy of such treatment regimens are determined in cell culture or experimental animals, including but not limited to determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (The dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly regulators that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are optionally used to formulate a dosage range for use in humans. Such capsid assembly regulators are preferably used in an amount within a circulating concentration range that includes the ED50 where toxicity is minimal. Optionally, the dosage varies within this range depending on the dosage form and route of administration employed.

[0255] Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims appended hereto. For example, it should be understood that it is within the scope of this application to use alternative methods recognized in the art and to modify the detection and / or reaction conditions by routine experimentation.

[0256] Example

[0257] This specification is further described in detail with reference to the following experimental examples. These examples are for illustrative purposes only and are not limiting unless otherwise stated. Therefore, this specification should not be interpreted as being limited to the following examples, but should be interpreted as including any and all changes shown by the teachings provided herein.

[0258] Example 1: Overview

[0259] PCLD and an important subset of ADPKD are caused by insufficient PC1 function, which is caused by loss-of-function mutations in the PKD1 gene itself or mutations in the endoplasmic reticulum (ER) gene responsible for maturation of PC1 from the ER to the surface of primary ciliated cells. Therefore, increasing the functional dosage of PC1 is expected to be able to treat, improve or prevent PCLD and ADPKD.

[0260] As described in Example 1, the studies described herein ("the present study") discovered two upstream open reading frames (uORFs) in the 5'-untranslated region (5'-UTR) of PKD1 messenger RNA (mRNA). The present study further found that these two uORFs reduced the translation efficiency of PKD1, while abrogating the uORFs by mutation or blocking the uORFs using antisense oligonucleotides (ASOs) significantly enhanced the translation of PKD1 mRNA and increased the protein level of PC1.

[0261] Specifically, the in vitro data described here show that when only 2 nucleotides of the PKD1 5'UTR were edited to abolish uORF translation, the steady-state expression of downstream genes increased by approximately 4-fold. This study also demonstrated that the use of antisense oligonucleotides (ASOs) to inhibit PKD1 uORF translation in human cells almost reproduced the expression fold changes evaluated by base editing, so this highly specific therapy is also highly achievable.

[0262] Since the two uORFs in the 5'-UTR of PKD1 have unique sequences, and methods targeting uORFs sequences (such as CRISPR or ASO) can be designed with extremely high specificity, the treatment of ADPKD or PCLD through uORFs is expected to achieve high specificity. Considering the fold change in PKD1 protein expression achieved by inhibiting uORFs, it is speculated that this degree of effect is sufficient to prevent cyst formation in at least some models or patients and is a safe long-term treatment. In addition, this approach is expected to be effective in all patients with isolated polycystic liver disease (symptomatic incidence is approximately 1:10,000-1:150,000) and a portion of ADPKD patients (total incidence is approximately 1:400-1:1000).

[0263] Example 1-1: Related information

[0264] Autosomal dominant polycystic kidney disease (ADPKD) has a prevalence of 1:400-1:1000 and results in renal failure in 5-10%. ADPKD is caused by pathogenic mutations in the PKD1 or PKD2 genes (encoding polycystin 1 (PC1) and polycystin 2 (PC2)) and is characterized by enlarged renal and hepatic cysts. The overlapping spectrum of renal and hepatic cyst phenotypes, ADPKD-ADPLD (also known as isolated polycystic liver disease or PCLD), is caused by mutations in genes required for PC1 maturation in the endoplasmic reticulum (ER): SEC63, PRKCSH, ALG8, ALG9, GANAB, SEC61B, DNAJB117. Truncating mutations in PKD1 that result in complete loss of PC1 function lead to the most rapid progression to renal failure. Nonetheless, patients with non-truncating PKD1 mutations still develop renal failure, and these and many ADPKD-ADPLD cases desperately need a treatment to halt the symptoms and complications of the relentless accumulation and expansion of renal and / or hepatic cysts. The late onset of renal failure in patients with non-truncating PKD1 mutations (approximately one-third of ADPKD) suggests that the encoded PC1 is at least partially functional. A significant subset of these missense mutant PC1 constructs matured inefficiently at the cell surface, suggesting that quantitative expression is critical. Interestingly, in mouse models of cystic kidneys with impaired ER maturation of Prkcsh or Sec63—a quantitative increase in PC1 production achieved by mice carrying three copies of the Pkd1 gene encoding the gene—was sufficient to allow enough PC1 to reach the cell surface to avoid cyst formation. Together, these findings suggest that, in cases where PC1 maturation is inefficient, increasing PC1 expression can successfully increase the amount of function that reaches the cell surface, thereby preventing cyst formation.

[0265] Investigations at the University of Texas Southwestern have proposed an approach to inhibit the microRNA that degrades PKD1 mRNA. Genetic mouse models have indeed demonstrated benefit from this approach with increased PC1 expression (Lakhia et al., Nat Comm. 2022).

[0266] However, the microRNA approach will target a large number of genes, and therapies based on this will be severely limited by off-target effects. For this reason, it is believed that targeting the specificity of the unique sequence of the PKD1 uORF, combined with a similar doubling (if not more) of PC1 protein expression prediction, will have similar (if not more) desired efficacy without the risks.

[0267] Example 1-2: PKD1 mRNA includes at least three upstream open reading frames (uORFs) in the 5'UTR

[0268] In calculating the translation probability of the identified uORFs, three potential uORFs in the 5'-untranslated region (5'UTR) of PKD1 were identified in the top 0.1% of all scored genome-wide uORFs. The sequences of these uORFs (uORF1, uORF2, uORF3) and the proposed uORF4 are listed in SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, respectively. Figure 2A Two ATG-initiated uORFs (uORF1 and uORF2) are shown. Figure 2B These uORFs and the predicted 5'UTR secondary structure are further illustrated.

[0269] The 5' untranslated region (5'UTR) of the messenger RNA (mRNA) sequence of many genes contains potential translation initiation sites that encode short or out-of-frame peptides from the gene of interest; these sites are called upstream open reading frames (uORFs). For some other genes whose uORFs were studied, translation of the uORF had a deleterious effect on the translation of the intended protein by reducing the number of ribosomes that reach the start codon of the protein sequence ( Figure 3A-3B ).

[0270] Example 1-3: Genetically abolishing the uORF in the 5'UTR of PKD1 significantly increases protein expression

[0271] refer to Figures 4A-4D , by incorporating the dual luciferase reporter gene ( Figure 4A ) in which the 5'UTR of Renilla luciferase was replaced with the wild-type human PKD1 5'UTR ("wt") or the human PKD1 5'UTR containing one or two single-base edits to abolish one or both of the uORF start sites ("ΔuORF1", "ΔuORF2", and "ΔuORF1&2") ( Figure 4B ) to prepare constructs. Cells were then transfected with the four constructs. Mutations that abolished the uORF did not alter mRNA expression or transfection efficiency ( Figure 4D However, this targeted editing resulted in a significant increase in luciferase protein expression by nearly 4-fold ( Figure 4C , “wt” vs. “1&2”).

[0272] Example 1-4: ASOs that inhibit uORF in PKD1 5'UTR significantly increase protein expression

[0273] Based on the sequence of the uORF and the sequence of the 5'UTR, two non-limiting examples of antisense oligonucleotides were designed: uORF1 ASO ("ASO1") (CAUGGCGGGCGCGGGG, SEQ ID NO: 158, complementary to the 5'UTR sequence before and including the AUG start codon of uORF1), and uORF2 ASO ("ASO2") (CAUGGCCCCGCCGUCC, SEQ ID NO: 159, complementary to the 5'UTR sequence before and including the AUG start codon of uORF2). Both ASOs include 2'-O-methylated modified bases and phosphorothioate (PS) linkages to increase resistance to degradation.

[0274] After transfection of cells expressing a luciferase reporter gene including the PKD1 wild-type 5'UTR (see "Examples 1-3" section), cells were cultured with one or two ASOs (ASO1, ASO2 or ASO1+2) or mismatched control (MM1, MM2) ASOs. Lysates were collected after 24 hours of treatment. Figure 5 , the luciferase expression in the cell lysates treated with uORF1 and uORF2 ASOs showed an approximately 3-fold increase compared to that in the cell lysates treated with the control group.

[0275] refer to Fig. 6A , wild-type epithelial cells were treated with 20 nm ASO and observed a 1.5-2 fold increase in PC1 steady-state expression levels compared to that seen with mismatched ASOs, without any significant changes in mRNA expression. This is consistent with the proposed mechanism by which uORF inhibition promotes translation without affecting transcripts. Increases in PC1 expression were observed after 12, 24, 48, and 96 h of treatment ( Figure 6B-6D ). Reference Fig. 6E , when the concentration of ASO was 10 nM or 20 nM, a strong upregulation of PC1 protein expression was observed.

[0276] Example 1-5: Design of ASOs for sterically blocking translation of mouse Pkd1 uORF

[0277] Reference Figure 8 , studies of the mouse Pkd1 mRNA sequence revealed two ATG-initiated potential uORFs (mouse uORF1 and mouse uORF2), which correspond to the two human PKD1 uORFs.

[0278] Reference Fig. 9, guided by published information (Liang et al., Nat Biotechnol 34, 875-880 (2016) and Liang et al. (Nucleic acids research 45, 9528-9546 (2017)), two ASOs that inhibit mouse uORF (mouse ASO1 and mouse ASO2), and two mismatch control ASOs (mouse MM-1 and mouse MM-2) were designed. Specifically, the ASOs (each 16 nucleotides in length and including 2'-O-methyl (2'-OMe) modification and phosphorothioate (PS) linkage) were designed to have the following sequences: mouse uORF-1 ASO (mouse ASO1): 5'mC*mA*mU*mG*mG*mU*mG*mC*mG*mG*mC*mA*mC*mG*mG*mG 3' (SEQ ID 3' (SEQ ID NO: 162), and mouse mismatch control 2 (mouse MM-2): 5'mC*mU*mU*mG*mC*mU*mG*mU*mG*mG*mG*mA*mC*mC*mG*mG 3' (SEQ ID NO: 163). NO: 163). In the ASOs of this paragraph, "m" indicates that the sugar group of these ASOs has a 2'-O-methyl (2'-OMe) modification, and "*" indicates that the linkage is a phosphorothioate (PS) linkage.

[0279] Two mouse ASOs and two control ASOs were tested in a mouse cell line containing an HA epitope tag at the C-terminus of Pkd1 to allow evaluation of C-terminal fragments. Fig. 10A and 10B Treatment of mouse cell lines with 20 nM mouse ASO1, ASO2, or a combination of both ASOs significantly increased the levels of mature PC1 protein in the cell lines.

[0280] The ASO was then tested in another mouse cell line whose PC1 protein contained the missense mutation R2216W, as well as a V5 epitope tag at the C-terminus of the same protein for detection. The mouse R2216W mutation corresponds to the R2220W mutation in the human PC1 protein, which is present in some ADPKD patients and has been found to affect the cleavage and maturation of the protein (Vujic et al., J Am Soc Nephrol. 2010 Jul; 21(7): 1097-102 and Krappitz et al., JASN 2023). Fig.11 As shown, even for the mutant PC1 R2216W protein that is resistant to cleavage and maturation, treatment with ASO significantly increased the level of mature PC1 protein (cleaved protein).

[0281] Example 2: (Hypothetical) ASOs that inhibit uORF3 and / or uORF4 increase PC1 protein levels

[0282] In addition to uORF1 and uORF2 described above, further analysis of the human PKD1 mRNA 5′-UTR sequence revealed two additional potential uORFs: uORF3 and uORF4, for which there is evidence of translation in at least one published ribosome profiling experiment ( Fig.12 ).

[0283] The ASOs are designed to inhibit uORF3 and uORF4. Specifically, the ASOs are designed to bind to human PKD1 mRNA molecules near or including the start codon of uORF3 or uORF4 (the start codons of uORF3 and uORF4 are both "CUG") and block the translation of one or both of these two uORFs. Testing of the ASOs (e.g., in human cell lines) has shown that these ASOs are able to increase the expression of PC1 protein and its mature form, similar to ASO1 / 2 that inhibits uORF1 / 2.

[0284] Example 3: (Hypothetical) ASOs that inhibit uORF in the Pkd1 5'UTR reduce the cystic phenotype in the ADPKD mouse model

[0285] Reference Figure 7 , knocking out one copy of Dnajb11 and Pkd1 during embryogenesis (Ksp-Cre is active in the distal glomerulus from mid-embryogenesis onwards) results in mice with a strong cystic phenotype.

[0286] Using this mouse disease model, we found that administration of a mouse uORF ASO significantly increased PC1 protein expression of the single remaining allele, and a two-fold increase was predicted to abolish the cystic phenotype in the mouse kidney.

[0287] Example 4: (Hypothetical) Assessment of CTG-initiated uORFs by Western Blot

[0288] Retinal pigment epithelial (RPE) cells were cultured and treated with vector, ASO3 (targeting uORF3), negative control mismatched ASO3 ("ASOMM3", an altered sequence that does not actually target uORF3), ASO1 (positive control), or ASO1+ASO3 for 24 or 96 hours, protein lysates were extracted from the cells, and the expression of PC1 was assessed by western blotting.

[0289] This experiment was repeated to compare the normalized luciferase expression of wild-type PKD1 5'UTR-Renilla luciferase constructs treated with vehicle, ASO3, ASOMM3, ASO1, or ASO1+ASO3.

[0290] The effect of blocking uORF3 on PC1 expression will be similar to the effect of blocking the other uORFs described herein.

[0291] Further experiments will modify the sequence of PKD1-5'UTR in our psiCHECK2 luciferase expression plasmid containing human PKD1 5'UTR to contain a combination of gene sequence modifications ΔuORF3, or ΔuORF4, and ΔuORF1 or ΔuORF2, similar to Figure 4B Modification of gene sequence The CTG start codon of uORF3 or uORF4 was changed to TTG to prevent the initiation of translation. Renilla: Firefly luciferase expression of gene modified constructs compared to wild-type construct (baseline) and delta-uORF1 construct (positive control based on the results shown).

[0292] Examples of implementation methods

[0293] In some embodiments, this description relates to the following non-limiting embodiments:

[0294] Embodiment 1: A method for treating, ameliorating and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof, comprising:

[0295] An effective amount of a compound that inhibits translation of a first upstream open reading frame (uORF), a second uORF, a third uORF, and / or a fourth uORF of the PKD1 gene is administered to the subject.

[0296] Embodiment 2: The method according to embodiment 1, wherein the method is a method for treating, ameliorating and / or preventing ADPKD in a subject, and wherein ADPKD is caused by or involves a PKD1 gene mutation in the subject.

[0297] Embodiment 3: A method according to embodiment 1, wherein the method is a method for treating, improving and / or preventing PCLD in a subject, and wherein PCLD is caused by or involves a germline mutation of a PKD1 gene, a PKD2 gene, a PRKCSH gene, a SEC63 gene, a GANAB gene, an ALG8 gene, an ALG9 gene, a SEC61B gene or a DNAJB11 gene in the subject.

[0298] Embodiment 4: The method according to embodiment 1-3, wherein the compound comprises:

[0299] A CRISPR component or an expression vector expressing the CRISPR component, the CRISPR component disrupting a genomic DNA sequence encoding the first uORF, the second uORF, the third uORF and / or the fourth uORF; or

[0300] An antisense oligonucleotide (ASO) or an expression vector expressing the ASO, wherein the ASO blocks the translation of the first uORF, the second uORF, the third uORF and / or the fourth uORF.

[0301] Embodiment 5: The method according to embodiment 4, wherein the compound comprises a CRISPR component or an expression vector expressing a CRISPR component, and wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF and / or the fourth uORF.

[0302] Embodiment 6: The method according to embodiment 4, wherein the compound comprises an ASO or an expression vector expressing an ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less from the start codon of the first uORF, the second uORF, the third uORF or the fourth uORF, such as extending to 4 nucleotides or less, extending to 3 nucleotides or less, extending to 2 nucleotides or less, extending to 1 nucleotide or less, reaching the boundary of the start codon, reaching 1 nucleotide or more of the start codon, reaching 2 nucleotides or more of the start codon, or reaching the entire start codon of the first uORF, the second uORF, the third uORF or the fourth uORF.

[0303] Embodiment 7: The method according to embodiment 4 or 6, wherein the length of the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer or 15 nucleotides or longer.

[0304] Embodiment 8: The method according to embodiments 4 and 6-7, wherein the length of the ASO is 30 nucleotides or shorter, such as 29 nucleotides or shorter, 28 nucleotides or shorter, 27 nucleotides or shorter, 26 nucleotides or shorter or 25 nucleotides or shorter.

[0305] Embodiment 9: The method according to embodiments 4 and 6-8, wherein at least one of the following applies:

[0306] (a) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 14-61;

[0307] (b) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 62-109;

[0308] (c) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 110-157;

[0309] (d) ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159).

[0310] Embodiment 10: The method according to embodiments 4 and 6-9, wherein the ASO comprises a modified base, a modified sugar group or a modified linkage.

[0311] Embodiment 11: The method of embodiment 10, wherein at least one of the following applies:

[0312] (a) the ASO comprises a modified sugar group, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group;

[0313] (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

[0314] Embodiment 12: The method according to embodiments 1-11, wherein the subject is a mammal, such as a human.

[0315] Embodiment 13: The method of embodiments 1-12, wherein the compound comprises an ASO or an expression vector expressing the ASO, and wherein the concentration of the ASO in the kidney or lung of the subject ranges from about 1 nm to about 100 nm.

[0316] Embodiment 14: A method for increasing PKD1 expression in a cell, comprising:

[0317] An effective amount of a compound that inhibits translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene is contacted with the cell.

[0318] Embodiment 15: The method according to embodiment 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.

[0319] Embodiment 16: The method according to any one of embodiments 14-15, wherein the cell is in a tissue or a subject.

[0320] Embodiment 17: The method according to any one of embodiments 14-16, wherein 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).

[0321] Embodiment 18: The method according to any one of embodiments 14-17, wherein the compound comprises:

[0322] A CRISPR component or an expression vector expressing the CRISPR component, the CRISPR component disrupting a genomic DNA sequence encoding the first uORF, the second uORF, the third uORF and / or the fourth uORF; or

[0323] An antisense oligonucleotide (ASO) or an expression vector expressing the ASO, wherein the ASO blocks the first uORF, the second uORF, the third uORF and / or the fourth uORF.

[0324] Embodiment 19: The method of embodiment 18, wherein the compound comprises a CRISPR component or an expression vector expressing a CRISPR component, and wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF and / or the fourth uORF.

[0325] Embodiment 20: A method according to embodiment 18, wherein the compound comprises an ASO or an expression vector expressing an ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less from the start codon of the first uORF, the second uORF, the third uORF or the fourth uORF, such as extending to 4 nucleotides or less, extending to 3 nucleotides or less, extending to 2 nucleotides or less, extending to 1 nucleotide or less, reaching the boundary of the start codon, reaching 1 nucleotide or more of the start codon, reaching 2 nucleotides or more of the start codon, or reaching the entire start codon of the first uORF, the second uORF, the third uORF or the fourth uORF.

[0326] Embodiment 21: A method according to embodiment 18 or embodiment 20, wherein the length of the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer or 15 nucleotides or longer.

[0327] Embodiment 22: A method according to any one of embodiments 18 and 20-21, wherein the length of the ASO is 30 nucleotides or shorter, such as 29 nucleotides or shorter, 28 nucleotides or shorter, 27 nucleotides or shorter, 26 nucleotides or shorter or 25 nucleotides or shorter.

[0328] Embodiment 23: The method according to any one of embodiments 18 and 20-22, wherein at least one of the following applies:

[0329] (a) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 14-61;

[0330] (b) the ASO is completely complementary to one of the sequences in SEQ ID NOs: 62-109;

[0331] (c) the ASO is completely complementary to one of the sequences in SEQ ID NOs: 110-157;

[0332] (d) ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158), the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159).

[0333] Embodiment 24: The method according to any one of embodiments 18 and 20-23, wherein the ASO comprises a modified base, a modified sugar group or a modified linkage.

[0334] Embodiment 25: The method of embodiment 24, wherein at least one of the following applies:

[0335] (a) the ASO comprises a modified sugar, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group;

[0336] (b) The ASO comprises a modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

[0337] Embodiment 26: The method according to any one of embodiments 14-24, wherein the compound comprises an ASO or an expression vector expressing an ASO, and wherein the concentration of the ASO contacted with the cell ranges from about 1 nm to about 100 nm.

[0338] The features of several embodiments are summarized above so that those skilled in the art can better understand the various aspects of the present disclosure. Those skilled in the art should recognize that they can easily use the present disclosure as a basis for designing or modifying other processes and structures to implement the same purposes and / or achieve the same advantages of the embodiments described herein. Those skilled in the art should also recognize that such equivalent structures do not depart from the spirit and scope of the present disclosure, and various changes, substitutions and modifications can be made to the present disclosure without departing from the spirit and scope of the present disclosure.

Claims

1. A method for treating, ameliorating and / or preventing autosomal dominant polycystic kidney disease (ADPKD) or polycystic liver disease (PCLD) in a subject in need thereof, comprising: An effective amount of a compound that inhibits translation of a first upstream open reading frame (uORF), a second uORF, a third uORF, and / or a fourth uORF of the PKD1 gene is administered to the subject. 2 . The method according to claim 1 , wherein the method is a method for treating, ameliorating and / or preventing ADPKD in a subject, and wherein the ADPKD is caused by or involves a PKD1 gene mutation in the subject.

3. The method of claim 1, wherein the method is a method for treating, ameliorating and / or preventing PCLD in a subject, and wherein the PCLD is caused by or involves a germline mutation of a PKD1 gene, a PKD2 gene, a PRKCSH gene, a SEC63 gene, a GANAB gene, an ALG8 gene, an ALG9 gene, a SEC61B gene or a DNAJB11 gene in the subject.

4. The method according to any one of claims 1 to 3, wherein the compound comprises: A CRISPR component or an expression vector expressing the CRISPR component, wherein the CRISPR component disrupts a genomic DNA sequence encoding the first uORF, the second uORF, the third uORF and / or the fourth uORF; or An antisense oligonucleotide (ASO) or an expression vector expressing the ASO, wherein the ASO blocks the translation of the first uORF, the second uORF, the third uORF and / or the fourth uORF.

5. The method of any one of claims 1 to 4, wherein the compound comprises the CRISPR component or an expression vector expressing the CRISPR component, and wherein the CRISPR component disrupts the start codon of the first uORF, the second uORF, the third uORF and / or the fourth uORF.

6. The method of claim 4, wherein the compound comprises the ASO or an expression vector expressing the ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less, such as 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, 1 nucleotide or less, to the boundary of the start codon, to 1 nucleotide or more of the start codon, to 2 nucleotides or more of the start codon, or to the entire start codon of the first uORF, the second uORF, the third uORF or the fourth uORF.

7. The method according to claim 4 or 6, wherein the ASO has a length of 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer or 15 nucleotides or longer.

8. The method according to any one of claims 4 and 6 to 7, wherein the ASO has a length of 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.

9. The method according to any one of claims 4 and 6 to 8, wherein at least one of the following applies: (a) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 14-61; (b) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 62-109; (c) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 110-157; (d) The ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158) and the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159).

10. The method according to any one of claims 4 and 6 to 9, wherein the ASO comprises a modified base, a modified sugar group or a modified linkage.

11. The method of claim 10, wherein at least one of the following applies: (a) the ASO comprises the modified sugar group, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group; (b) the ASO comprises the modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

12. The method according to any one of claims 1 to 11, wherein the subject is a mammal, such as a human.

13. The method of any one of claims 1 to 12, wherein the compound comprises the ASO or an expression vector expressing the ASO, and wherein the concentration of the ASO in the kidney or lung of the subject ranges from about 1 nm to about 100 nm.

14. A method for increasing PKD1 expression in a cell, comprising: An effective amount of a compound that inhibits translation of the first upstream open reading frame (uORF), the second uORF, the third uORF, and / or the fourth uORF of the PKD1 gene is contacted with the cell.

15. The method of claim 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.

16. The method of any one of claims 14 to 15, wherein the cell is in a tissue or a subject.

17. The method of any one of claims 14 to 16, wherein the cell is a kidney cell in a subject diagnosed with autosomal dominant polycystic kidney disease (ADPKD) or a hepatocyte in a subject diagnosed with polycystic liver disease (PCLD).

18. The method according to any one of claims 14 to 17, wherein the compound comprises: A CRISPR component or an expression vector expressing the CRISPR component, wherein the CRISPR component disrupts a genomic DNA sequence encoding the first uORF, the second uORF, the third uORF and / or the fourth uORF; or An antisense oligonucleotide (ASO) or an expression vector expressing the ASO, wherein the ASO blocks the first uORF, the second uORF, the third uORF and / or the fourth uORF.

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

20. The method of claim 18, wherein the compound comprises the ASO or an expression vector expressing the ASO, and wherein the portion of the PKD1 mRNA complementary to the ASO extends to 5 nucleotides or less, such as 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, 1 nucleotide or less, to the boundary of the start codon, to 1 nucleotide or more of the start codon, to 2 nucleotides or more of the start codon, or to the entire start codon of the first uORF, the second uORF, the third uORF, or the fourth uORF.

21. The method of claim 18 or claim 20, wherein the ASO is 10 nucleotides or longer, such as 11 nucleotides or longer, 12 nucleotides or longer, 13 nucleotides or longer, 14 nucleotides or longer or 15 nucleotides or longer in length.

22. The method according to any one of claims 18 and 20-21, wherein the ASO is 30 nucleotides or shorter, such as 29 nucleotides or shorter, 28 nucleotides or shorter, 27 nucleotides or shorter, 26 nucleotides or shorter or 25 nucleotides or shorter in length.

23. The method according to any one of claims 18 and 20-22, wherein at least one of the following applies: (a) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 14-61; (b) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 62-109; (c) the ASO is completely complementary to a sequence listed in SEQ ID NOs: 110-157; (d) The ASO comprises the nucleotide sequence CAUGGCGGGCGCGGGG (SEQ ID NO: 158) and the nucleotide sequence CAUGGCCCCGCCGUCC (SEQ ID NO: 159).

24. The method of any one of claims 18 and 20-23, wherein the ASO comprises a modified base, a modified sugar group or a modified linkage.

25. The method of claim 24, wherein at least one of the following applies: (a) the ASO comprises a modified sugar, and the modified sugar group comprises a 2'-O-methylated modified sugar group, such as a 2'-O-methylated modified ribose group; (b) the ASO comprises the modified linkage, and the modified linkage comprises a phosphorothioate (PS) linkage.

26. The method of any one of claims 14 to 24, wherein the compound comprises the ASO or an expression vector expressing the ASO, and wherein the concentration of the ASO contacted with the cell ranges from about 1 nm to about 100 nm.

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