Gene therapy for Dent's disease

Abstract

The present invention includes methods and compositions useful for treating Dent disease in a subject in need thereof. The disclosed invention also includes a mouse model useful for studying Dent disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 193,212, filed May 26, 2021, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing An ASCII text file named "205286_7010WO1SequenceListing" containing 29 kilobytes, created on May 20, 2022, is incorporated by reference in its entirety into this specification. [Background technology]

[0003] 2. Background of the Invention Dent's disease is a chronic kidney disorder characterized by abnormally high amounts of protein and excess calcium in the urine. Dent's disease is caused by a genetic mutation that reduces the ability of cells in the proximal renal tubules to reabsorb nutrients, water and other substances filtered from the bloodstream. Clinical symptoms of Dent's disease appear in early childhood and worsen over time. The kidney dysfunction that causes Dent's disease gradually damages kidney cells, eventually causing calcification of kidney tissue, kidney stones, abdominal pain, recurrent urinary tract infections, and a wide range of symptoms from chronic kidney disease and kidney failure.

[0004] Genetically, Dent disease is caused by loss-of-function mutations in either the CLCN5 or OCRL1 genes, which divides the disease into two types. Type 1 Dent disease is characterized by mutations in the CLCN5 gene, while type 2 Dent disease involves mutations in OCRL1. Both genes are X-linked recessive, so that the majority of patients are male, but women may be asymptomatic "carriers" and suffer from mild hypercalciuria due to random X-chromosome inactivation. Type 1 Dent disease is common in around 60% of all cases, type 2 in 15% of cases, and the remaining 25% are of unknown etiology. Type 2 Dent disease is often accompanied by mild intellectual disability, hypotension, and mild cataracts. Currently, there are only supportive treatments for Dent disease, none of which target the cause of the disease. Severe cases are often treated by kidney transplantation, but such a strategy requires identification of a suitable donor, invasive surgery, and life-long immunosuppression to delay rejection.

[0005] Thus, there is a need for a therapy that corrects the mutated gene that causes Dent's disease in order to restore normal kidney function. The present invention addresses these needs. Summary of the Invention

[0006] As described herein, the present invention relates to methods and compositions useful for treating Dent disease type 1 in a subject in need thereof. The disclosed invention also includes a mouse model useful for studying Dent disease.

[0007] Thus, in one aspect, the present invention includes a method for treating Dent's disease in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid vector encoding a CLCN5 protein, thereby treating the disease.

[0008] In certain aspects, the nucleic acid vector is a lentiviral vector.

[0009] In certain embodiments, the nucleic acid vector is operably linked to a promoter that drives expression of a CLCN5 protein.

[0010] In certain aspects, the promoter is a constitutive promoter.

[0011] In one particular embodiment, the promoter is the EF-1α promoter.

[0012] In certain aspects, the promoter is a tissue-specific promoter.

[0013] In certain aspects, the tissue-specific promoter is specific to renal tubular proximal cells.

[0014] In certain embodiments, the tissue-specific promoter is selected from the group consisting of Npt2a and Sgt12.

[0015] In certain embodiments, the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1.

[0016] In certain embodiments, administration is locally delivered to the kidney.

[0017] In certain embodiments, localized renal administration is delivered by retrograde ureteral injection.

[0018] In another aspect, the invention includes a method for correcting a mutation in a CLCN5 gene in a cell, comprising contacting the cell with a nucleic acid vector encoding a functional CLCN5 protein.

[0019] In certain aspects, the nucleic acid vector is a lentiviral vector.

[0020] In certain embodiments, the nucleic acid vector is operably linked to a promoter that drives expression of a CLCN5 protein.

[0021] In certain aspects, the promoter is a constitutive promoter.

[0022] In one particular embodiment, the promoter is the EF-1α promoter.

[0023] In certain aspects, the promoter is a tissue-specific promoter.

[0024] In certain aspects, the tissue-specific promoter is specific to renal tubular proximal cells.

[0025] In certain embodiments, the tissue-specific promoter is selected from the group consisting of Npt2a and Sgt12.

[0026] In certain preferred embodiments, the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid vector encoding a CLCN5 protein and a pharma- ceutically acceptable carrier.

[0028] In certain aspects, the nucleic acid vector is a lentiviral vector.

[0029] In certain embodiments, the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1.

[0030] In another aspect, the invention includes a mouse model of Dent's disease type 1, wherein the mouse comprises one or more mutations in the mouse CLCN5 gene.

[0031] In certain embodiments, the one or more mutations are deletions.

[0032] In certain embodiments, the deletion affects exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11 of the CLCN5 gene.

[0033] In certain embodiments, the one or more CLCN5 mutations result in a non-functional CLCN5 protein.

[0034] In certain embodiments, breeding of laboratory animals requires sires and dams of different strains.

[0035] In certain embodiments, the female parent is heterozygous for a CLCN5 mutation and the male parent is wild type.

[0036] In certain embodiments, the sire is of an FVB background.

[0037] In certain embodiments, the dam is of a C57BL / 6 background. [Brief description of the drawings]

[0038] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0039] [Figure 1] 1A-1B are diagrams showing the overall mutation profile of the CLCN5 gene in Dent's disease. FIG. 1A is a diagram of the CLCN5 gene showing the location and type of known mutations. FIG. 1B is a chart showing the frequency of various mutations. [Diagram 2] Figures 2A-2B are schematics showing the strategy to generate a Dent's disease mouse model via deletion of CLCN5. Figure 2A shows the location (arrows) of the ends of the deleted region spanning exons 3-11. Figure 2B shows the sequence of the completed mutant demonstrating successful deletion of the targeted region. [Diagram 3] Figures 3A-3B illustrate the breeding strategy required to generate CLCN5 knockout mice. Figure 3A illustrates that the expected Mendelian ratio of normal and knockout mice is 50 / 50, however, when C57BL / 6 female carriers were bred with normal males of the same strain, far fewer knockout male pups than expected were born, suggesting embryonic lethality. Figure 3B illustrates the mixed C57BL / 6 and FVB background breeding strategy required to obtain the expected ratio of knockout, heterozygous (carrier) and wild-type pups. [Figure 4] FIG. 4 illustrates that CLCN5 knockout mice do not produce detectable levels of CLCN5 mRNA or protein. [Diagram 5] Figure 5 illustrates that CLCN5 knockout mice secrete dramatically more albumin into the urine than wild-type mice, as assayed by SDS-PAGE. Three concentrations (50 μg, 100 μg, and 200 μg) from two knockout and two wild-type mice were separated on the gel. The box indicates a band at approximately 66 kDa (presumably albumin). [Figure 6] FIG. 6 illustrates studies demonstrating higher urinary protein secretion in CLCN5 mutant mice as assayed by Western blotting for albumin (left) and vitamin D binding protein (right). [Figure 7] FIG. 7 is a diagram showing the design of lentiviral vectors for expression of hCLCN5. [Figure 8] FIG. 8 illustrates that CLCN5 lentiviral vectors can induce CLCN5 expression in transduced cells as measured by RT-qPCR (left) and Western blotting (right). [Figure 9]Figures 9A-9B illustrate the delivery of CLCN5 lentiviral vectors via retrograde ureteral injection. Figure 9A is a schematic of retrograde ureteral injection (left) and a photomicrograph of a well-positioned ureter and kidney during the injection procedure. Figure 9B is a fluorescent photomicrograph of kidney tissue from a mouse injected with GFP-expressing lentivirus one week earlier (right) or an untreated control mouse (left). [Figure 10] FIG. 10 is a diagram of the in vivo study set-up in which CLCN5 knockout mice were treated with a CLCN5-expressing lentiviral vector delivered via retrograde ureteral injection. [Figure 11] FIG. 11 illustrates that treatment of mutant mice with CLCN5 lentivirus significantly reduces urinary protein as assessed by SDS-PAGE. [Figure 12] Figure 12 illustrates the reduction of specific urinary proteins in lentivirus-treated knockout mice. Studies evaluated albumin (left) or vitamin D binding protein (DBP, right). [Figure 13] FIG. 13 illustrates the reduction of CC16 protein in lentivirus-treated mice. [Figure 14] FIG. 14 illustrates that the reduction in urinary protein in mutant mice induced by lentiviral therapy was sustainable up to two months post-injection as assessed by SDS-Page (left), while untreated mutants did not demonstrate any reduction in protein levels (right). [Figure 15] FIG. 15 illustrates urine volumes (top) and protein levels (middle) and calcium ion levels (bottom) from CLCN5 lentivirus-treated knockout mice, untreated control or mice that received GFP control lentivirus. [Figure 16A]Figures 16A-16E illustrate the generation and characterization of CLCN5 knockout mice. Figure 16A. Gene structure of mouse CLCN5 and sgRNA used to delete the 26 kilobp region. Figure 16B. RT-PCR confirmation of lack of CLCN5 mRNA expression in mutant mouse kidneys. Two normal and two mutant male mice were analyzed. RT-PCR products using RNA template from normal kidney without reverse transcription. Primers were specific for mouse CLCN5 cDNA. Figure 16C. Western blotting analysis of CLCN5 protein in kidney tissues of wild-type and mutant mice. Figure 16D. SDS-PAGE analysis of urinary proteins of wild-type and mutant males. * indicates the 61 kDa protein band observed in urine samples from mutant mice, but not from wild-type mice. Figure 16E. Western blotting analysis of urinary proteins from normal and mutant mice. Sample order for CC10 was re-aligned to match that of Alb and DBP. Alb: Albumin; DBP: Vitamin D Binding Protein; CC10: Clara Cells 10 KDa Secreted Protein. For (Figures 16D and 16E), equal volumes of urine samples were analyzed, with each lane containing urine samples from a different mouse. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 16E] See legend to Figure 16A. [Figure 17]Figures 17A-17E illustrate the expression of human CLCN5 in the kidneys of mutant mice. Figure 17A. Components of human CLCN5-expressing lentiviral vector. Figure 17B. Detection of mRNA expression in HEK293T cells by RT-qPCR. CLCN5-expressing and GFP-expressing lentiviral vectors (10 ng p24) were transduced into 2.5x104 HEK293T cells. 48 hours after transduction, CLCN5 expression was detected by qRT-PCR using primers hCLCN5-F and hCLCN5-R (see Table 1 for sequences). The primers were specific for the codon-optimized human CLCN5 mRNA expressed from the lentiviral vector and failed to detect endogenous CLCN5 mRNA. *** indicates p<0.0001 (t-test). Figure 17C. Western blotting detection of CLCN5 protein in transduced renal proximal tubule cells. CLCN5-expressing lentiviral vector (28ng p24) was transduced into 2.5x105 renal proximal tubule cells isolated from wild-type and mutant mice. Western blotting was performed 72 hours after transduction. Figure 17D. Detection of CLCN5 protein by Western blotting 2 weeks after CLCN5 LV was delivered to the kidney of mutant mice. Figure 17E. Detection of CLCN5 protein expression by immunofluorescence 2 weeks after CLCN5 LV was delivered to the kidney of mutant mice. FITC and Alex-594 conjugated secondary antibodies were used to detect CLCN5 in wild-type and mutant mice, respectively. [Figure 18-1] Figures 18A-18E illustrate the therapeutic effect of CLCN5 gene therapy. Figure 18A. Immunofluorescence analysis of megalin expression in mutant mice with and without CLCN5 LV delivery. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and shown in blue. [Figure 18-2]Figures 18A-18E illustrate the therapeutic effect of CLCN5 gene therapy. Figure 18B. Quantitative analysis of mean fluorescence intensity of tubules by ImageJ. Figure 18C. SDS-PAGE analysis of urinary proteins after lentiviral vector delivery to both kidneys of mutant mice. Figure 18D. Western blotting detection of urinary marker proteins before and after CLCN5 LV injection into the left kidney of mutant mice. Figure 18E. Western blotting detection of urinary marker proteins before and after gene delivery to both kidneys of mutant mice. For (Figures 18C-18E), urine samples were collected one month after viral vector injection. Equal urine volumes were analyzed per sample. Each lane contained samples from different mice. [Figure 19A] Figures 19A-19B illustrate that the therapeutic effect lasted up to 4 months after gene delivery. Figure 19A. Diuresis, urinary protein and urinary calcium levels at various time points after gene therapy. Group sizes are indicated by n. *** indicates P<0.0001 when comparing mice with both kidneys treated with ZsGreen LV treated mice (two-tailed t-test). [Figure 19B] Figures 19A-19B illustrate that the therapeutic effect lasted for up to 4 months after gene delivery. Figure 19B. Western blotting analysis of urinary marker proteins at various time points after CLCN5 gene delivery. Equal volumes of urine samples from mice with one kidney treated were loaded in each lane. [Figure 20A]Figures 20A-20F illustrate that delivery of a second dose of LV suggests the involvement of an immune response. Figure 20A. Scheme of the experiment. Closed triangles indicate the time for evaluation of therapeutic efficacy. Figure 20B. SDS-PAGE analysis of urinary proteins. All mice were male mutants. Mouse #6 was a naive mouse that received the first dose of viral vector, and mice #1-5 were male mutant mice that received a second CLCN5 LV dose 5 months after receiving the first dose. Figure 20B: Before viral injection; Figure 20A: After viral injection. Figure 20C. Effect of the first and second doses of viral injection on diuresis (left), urinary protein (center) and urinary calcium (right) excretion. Data were from the same five mice that received the first and second doses. *: P<0.05 (2-way ANOVA followed by Bonferroni post-hoc test). Figure 20D. Detection of vector genomic DNA using qPCR after the first and second vector injections. Figure 20E. Detection of hCLCN5 mRNA expression using RT-qPCR after the first and second vector injections. Figure 20F. Detection of CLCN5 protein expression using Western blotting after the first and second vector injections. The same mice were analyzed in panels Figure 20B, Figure 20D, Figure 20E and Figure 20F. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 20E] See legend to Figure 20A. [Figure 20F] See legend to Figure 20A. [Figure 21] Figure 21 illustrates confirmation of CLCN5 gene knockout by DNA sequencing. The sequence above the horizontal arrow was deleted in three founder females (no. 6, no. 20, and no. 34). The sgRNA target sequences (underlined) in intron 2 and exon 12 are shown. The PAM is in green. The reverse primer in exon 12 was used for sequencing. The junction between intron 2 and exon 12 is indicated by a vertical arrow. [Figure 22] Figure 22 illustrates that fewer CLCN5 mutant males were obtained than expected. *** indicates p<0.0001 by Fisher's exact test. [Diagram 23] Figure 23 illustrates the reduction of urinary protein after delivery of a CLCN5-expressing lentiviral vector to the left kidney of mutant mice. Urine samples were collected one month after viral vector injection. The same urine volume was analyzed per sample. [Figure 24] Figure 24 illustrates that urinary protein levels returned to pre-treatment levels 4 months after gene delivery. The same urine volume was analyzed per sample. B: Pre-treatment; 1m-4m: 1, 2, 3 or 4 months after gene delivery. Samples from two representative mice are shown. [Diagram 25] Figures 25A-25B illustrate the generation and characterization of Clcn5 knockout mice. Figure 25A. Comparison of urine volume, urine calcium and urine protein in female mice. Wild-type, heterozygous and homozygous mutant mice were 81 days old. *, ** and *** indicate p<0.05, p<0.01 and p<0.001 between the indicated groups (one-way ANOVA followed by Tukey's multiple comparison test). Figure 25B. Comparison of urine volume, urine calcium and urine protein in male mice. Urine samples were collected from mice aged 2-2.5 months. *** indicates p<0.0001 between wild-type and mutant mice (two-tailed unpaired t-test). [Figure 26]Figures 26A-26B illustrate the delivery of LV vectors to mouse kidneys by retrograde ureteral injection. Figure 26A. Detection of GFP protein expression in mouse kidneys 2 weeks after GFP LV delivery by retrograde ureteral injection. Mice were 6-month-old wild type and received GFP LV injections in both kidneys. GFP expression was detected by immunofluorescence (shown in red). The inset was a magnified view of a GFP-positive tubule. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI, shown in blue). Figure 26B. Detection of GFP LV DNA in various organs by qPCR 2 weeks after GFP LV delivery. Genomic DNA samples isolated from various organs were used as templates in qPCR to detect GFP DNA. Mouse No. 1 was the same mouse shown in Figure 26A. Mouse No. 2, No. 3, and No. 4 were male Clcn5 mutant mice that received GFP LV injections 10 months after CLCN5 LV injections. All mice were euthanized 2 weeks after GFP LV injection. The dashed line indicates the limit of detection. [Figure 27] Figures 27A-27C illustrate that CLCN5 LV restored CLCN5 expression in the kidneys of mutant mice. Figure 27A. Detection of CLCN5 protein by immunofluorescence in wild-type kidneys. The inset shows relatively weak CLCN5 expression in the glomeruli marked with an asterisk. Figure 27B. Undetectable CLCN5 protein in the kidneys of mutant mice without CLCN5 LV injection. Figure 27C. Detection of CLCN5 protein in the kidneys of mutant mice two weeks after CLCN5 LV injection. The two half images were from two kidneys that received injections with different CLCN5 expression levels. [Figure 28]Figures 28A-28C illustrate the therapeutic effect of CLCN5 LV gene therapy. Figure 28A. Effect of CLCN5 LV delivery on diuresis in mutant mice. Figure 28B. Effect of CLCN5 LV delivery on urinary calcium in mutant mice. Figure 28C. Effect of CLCN5 LV delivery on urinary protein in mutant mice. For (Figures 28A-28C), all mutant mice received 280 ng of p24 CLCN5 or ZsGreen LV into the left kidney at 87 days of age. Data from each mouse is presented. The first reference point indicates the time of LV injection, and pre-treatment urinary parameters were from urine samples collected 37 days before LV injection. Post-treatment data showed urinary parameters from urine samples collected at the indicated ages. The dashed lines indicate the values ​​for wild-type male mice presented in the previous study presented here. *** indicates p<0.001 compared to pre-treatment values ​​(one-way ANOVA followed by Tukey's multiple comparison test). [Figure 29] Figures 29A-29C illustrate the therapeutic effect of delivering CLCN5 LV to both kidneys. Figure 29A. Effect of CLCN5 LV delivery on diuresis in mutant mice. Age-matched mutant mice were injected with CLCN5 LV or ZsGreen LV into both kidneys. For visualization, data from three of the five pairs are presented here, and data from the other two pairs are presented in Figure 33. Figure 29B. Effect of CLCN5 LV delivery on urinary calcium in mutant mice. Figure 29C. Effect of CLCN5 LV delivery on urinary protein in mutant mice. All mutant mice received 280 ng of p24 CLCN5 or ZsGreen LV into both kidneys at the age of the first data point. Data from each mouse are presented. Pre-treatment urine samples were collected 27 days prior to LV injection. The first reference point age for each mouse was the age of injection. Post-treatment urine samples were collected at the indicated ages. The dashed line indicates the value for wild-type male mice presented in the previous study of this disclosure. [Diagram 30]Figure 30 illustrates DNA sequencing analysis of predicted off-targets in Clcn5 gene knockout mice. The protospacer adjacent motifs (or reverse complementary sequences) are underlined in red, and the target sequences are underlined in black. Off1, Off2 and Off3 were off-targets for sgRNA1, sgRNA2 and sgRNA3, respectively. The last image was the only off-target on a protein-coding gene. Also, four off-targets on the X chromosome were labeled. [Diagram 31] Figures 31A-31C illustrate the effect of delivering CLCN5 LV to the left kidney. Figure 31A. Urine volume. Figure 31B. Urine calcium. Figure 31C. Urine protein. CLCN5 LV (280 ng p24) injections were performed on the first baseline day for each mouse. Urine was collected 37 days prior to LV injection. The second, third and fourth data points indicated the actual time that the urine sample was collected. [Diagram 32] Figures 32A-32C illustrate that age did not significantly affect urinary parameters of mutant mice. Figure 32A. Urine volume. Figure 32B. Urine calcium. Figure 32C. Urine protein. Each reference point was from a different male mutant mouse. Dashed lines indicate 95% confidence intervals. [Diagram 33] Figure 33 illustrates CLCN5 gene therapy for diuresis. Two of the five age-matched pairs are presented here for visualization. The other three pairs are shown in Figure 6A. Both kidneys were treated. [Diagram 34] Figures 34A-34C illustrate the effect of delivering CLCN5 LV to both kidneys. Figure 34A. Urine volume. Figure 34B. Urine calcium. Figure 34C. Urine protein. CLCN5 LV (280 ng p24 / kidney) injections were performed on the first baseline day for each mouse. Urine was collected 7 days prior to LV injection. The second, third, fourth and fifth data points indicated the chronological age at which urine samples were collected. [Diagram 35]Figure 35 depicts the detection of GFP protein by immunofluorescence in mouse kidneys with and without CLCN5 LV injection. Naïve mice were 6-month-old wild-type mice that received GFP LV injection without CLCN5 LV prior injection. The other three mice (CLCN5-LV, GFP-LV, no. 2-no. 4) were mutant mice that received GFP LV injection 10 months after CLCN5 LV injection. Mice were euthanized 2 weeks after GFP LV injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0041] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0042] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value such as an amount, temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, since such variations are reasonable in performing the disclosed methods.

[0044] "Biomarker" or "marker" as used herein generally refers to a nucleic acid molecule, clinical indicator, protein, or other analyte associated with a disease. In certain embodiments, a nucleic acid biomarker is an indicator of the presence in a sample of a pathogenic microorganism, including but not limited to viruses, viroids, bacteria, fungi, parasitic helminths, and protozoa. In various embodiments, a marker is differentially present in a biological sample obtained from a subject having or at risk of developing a disease (e.g., an infectious disease) compared to a reference. A marker is differentially present when the mean or median level of the biomarker present in the sample is statistically different from the level present in the reference. The reference level can be, for example, a level present in an environmental sample obtained from a clean or uncontaminated source. The reference level can be, for example, a level present in a sample obtained from a healthy control subject, or a level obtained from the subject at an earlier time point, i.e., before treatment. Common tests for statistical significance include, among others, t-tests, analysis of variance (ANOVA), Kruskal-Wallis, Wilcoxon, Mann-Whitney, and odds ratios. Biomarkers, alone or in combination, provide a measure of the relative likelihood that a subject belongs to a phenotypic state of interest. Differential presence of the markers of the invention in a subject sample can be useful in characterizing a subject as having or at risk of developing a disease (e.g., an infectious disease), for determining a subject's prognosis, for assessing treatment efficacy, or for selecting a treatment regimen.

[0045] By "agent" is meant any nucleic acid molecule, small molecule compound, antibody or polypeptide, or fragment thereof.

[0046] By "change" or "change" is meant an increase or decrease. The change may be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or even as much as 40%, 50%, 60%, or even 70%, 75%, 80%, 90% or 100%.

[0047] By "biological sample" is meant any tissue, cell, fluid, or other material derived from a living organism.

[0048] By "capture reagent" is meant a reagent that specifically binds to a nucleic acid molecule or a polypeptide in order to select or isolate the nucleic acid molecule or polypeptide.

[0049] As used herein, the terms "determining," "assessing," "assaying," "measuring," and "detecting" refer to both quantitative and qualitative determinations, and thus the term "determining" is used interchangeably herein with "assaying," "measuring," and the like. When a quantitative determination is intended, the phrase "determining the amount" of an analyte, etc. is used. When a qualitative and / or quantitative determination is intended, the phrase "determining the level" of an analyte or "detecting" an analyte is used.

[0050] "Detectable moiety" refers to a composition that, when linked to a molecule of interest, makes the molecule detectable through spectroscopic, photochemical, biochemical, immunochemical or chemical means.For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens.

[0051] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not remedied. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0052] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0053] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0054] By "fragment" is meant a portion of a nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.

[0055] "Homology" as used herein refers to the subunit sequence identity between two polymer molecules, for example, between two nucleic acid molecules, for example, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of positions that are matched or homologous; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits length) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0056] "Hybridization" means hydrogen bonding, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleotides that pair through the formation of hydrogen bonds.

[0057] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that are matched or identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0058] As used herein, "instruction manual" includes publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instruction manual of the kit of the present invention may be, for example, attached to a container containing the nucleic acid, peptide, and / or composition of the present invention, or may be shipped together with the container containing the nucleic acid, peptide, and / or composition. Alternatively, the instruction manual may be shipped separately from the container, with the intention that the instruction manual and the compound are used jointly by the recipient.

[0059] The terms "isolated," "purified," or "biologically pure" refer to a material that has been removed to various degrees from components that normally accompany it when found in its native state. "Isolate" refers to some degree of separation from the original source or environment. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein has been sufficiently freed from other materials such that any impurities do not substantially affect the biological properties of the protein or cause other deleterious events. That is, the nucleic acids or peptides of the invention are purified if cellular material, viral material, or culture medium, when produced by recombinant DNA technology, or chemical precursors or other chemicals, when chemically synthesized, have been substantially removed. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, the different modifications may result in different isolated proteins, which can be purified separately.

[0060] By "marker profile" is meant a characterization of the signal, level, expression or expression level of two or more markers (eg, polynucleotides).

[0061] The term "microorganism" refers to any organism that falls within the commonly used term "microbiology," including but not limited to bacteria, viruses, fungi, and parasites.

[0062] The term "microarray" refers to a collection of nucleic acid probes immobilized on a substrate. As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides or modified nucleotides and their polymers in single-stranded or double-stranded form. The term encompasses synthetic, naturally occurring and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that specifically binds to a target nucleic acid (e.g., a nucleic acid biomarker). Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide that has "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0063] The term "modulating" as used herein means to mediate a detectable increase or decrease in the level of a response in a subject, compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0064] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0065] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0066] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein that contains two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0067] "Reference" refers to a standard of comparison. As will be clear to those skilled in the art, a suitable reference is one for determining the effect of an element when the element is changed. In one aspect, the level of target nucleic acid molecule present in a sample can be compared with the level of target nucleic acid molecule present in a clean or uncontaminated sample. For example, the level of target nucleic acid molecule present in a sample can be compared with the level of target nucleic acid molecule present in corresponding healthy cells or tissues or diseased cells or tissues (e.g., cells or tissues from a subject with a disease, disorder or condition).

[0068] As used herein, the term "sample" includes biological samples such as any tissue, cell, fluid, or other biological material.

[0069] By "specifically binds" is meant a compound (e.g., a nucleic acid probe or primer) that recognizes and binds to a molecule (e.g., a nucleic acid biomarker) but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample.

[0070] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95%, 96%, 97%, 98% or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0071] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, the BLAST program may be used, e.g., glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. -3 ~e -100 A probability score between indicates closely related sequences.

[0072] The term "substantially a microbial hybridization signature" is a relative term and refers to a hybridization signature that indicates the presence of more microorganisms in the tumor sample than in the reference sample. The term "substantially no microbial hybridization signature" is a relative term and refers to a hybridization signature that indicates the presence of fewer microorganisms in the reference sample than in the tumor sample.

[0073] "Subject" means a mammal, including but not limited to a human or a non-human mammal, such as a cow, horse, dog, sheep, cat, mouse, or monkey. The term "subject" can refer to an animal (e.g., a patient) who is the object of treatment, observation, or experiment.

[0074] "Target nucleic acid molecule" refers to a polynucleotide to be analyzed. Such a polynucleotide may be the sense or antisense strand of a target sequence. The term "target nucleic acid molecule" also refers to an amplicon of the original target sequence. In various embodiments, the target nucleic acid molecule is one or more nucleic acid biomarkers.

[0075] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0076] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0077] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms. Although not exclusive, it will be recognized that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0078] The term "Dent disease" or "Dent's disease" as used herein refers to an X-linked renal syndrome of low molecular weight proteinuria, hypercalciuria, aminoaciduria, and hypophosphatemia caused by mutational defects in the genes encoding the CLCN5 and / or OCRL1 proteins that result in partial or complete loss of function of these genes. Loss of CLCN5 is associated with Dent disease type 1, while loss of OCRL1 is associated with Dent disease type 2.

[0079] Ranges: Throughout this disclosure, various aspects of the invention can be presented in the form of a range. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0080] explanation The present invention is based on the observation described herein that genetic abnormalities resulting in clinical disorders known as Dent disease can be treated by providing a nucleic acid vector encoding a functional replacement for the abnormal gene. The present invention also includes a mouse model of Dent disease, in which expression of Dent disease-related genes is knocked out in mutant mice, which is useful for studying Dent disease and developing treatments for the disease. Thus, in one aspect, the present invention includes a method for treating Dent disease in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid vector encoding a CLCN5 protein, thereby treating the disease. In another aspect, the disclosed invention includes a method for correcting a mutation in the CLCN5 gene in a cell, comprising contacting the cell with a nucleic acid vector encoding a functional CLCN5 protein.

[0081] Dent's disease Dent's disease is a kidney disorder characterized by the secretion of large amounts of small proteins and calcium ions into the urine, nephrocalcinosis, kidney stones, and chronic kidney disease. Progressive forms of the disease can result in kidney failure. Dent's disease is X-linked, and as a result, most patients are male; however, heterozygous females may suffer from milder forms of the disease, possibly due to random X inactivation in renal tissue. Symptoms of Dent's disease usually appear during early childhood; however, milder cases may remain undetected until adulthood. In some cases, the disorder gradually worsens over time, leading to chronic kidney disease and renal failure, typically by age 30-50.

[0082] Dent disease is further classified into two types. Type 1 Dent disease is characterized exclusively by the renal symptoms mentioned above, while type 2 Dent disease is characterized by the same renal symptoms, but also usually accompanied by other developmental disorders, including mild intellectual disability, eye lesions, or decreased muscle tone (hypotonia). Type 1 Dent disease is caused by mutations in the CLCN5 gene, while type 2 Dent disease is caused by mutations in the OCRL1 gene, both of which are located on the X chromosome. These mutations can be hereditary or occur randomly without any prior family history.

[0083] The CLCN5 gene encodes a voltage-dependent chloride channel of the chloride channel (CLC) family. CLCN5 is most highly expressed in renal proximal tubule cells, which normally reabsorb proteins that pass through the glomerular filter. A number of different mutations in CLCN5 have been observed in association with Dent's disease, all of which result in the loss of CLCN5 protein expression or the expression of a non-functional protein.

[0084] Current treatment for Dent's disease consists of supportive care for individual specific symptoms, which does not address the underlying genetic abnormality.Therefore, in certain embodiments, the present invention comprises the method for treating Dent's disease, comprising providing affected tissue with a functional copy of CLCN5 gene and CLCN5 protein.In certain embodiments, CLCN5 protein is delivered via the nucleic acid vector that codes for CLCN5 protein.

[0085] Gene transfer systems and lentiviral vectors Gene transfer systems, such as those described in the present invention, rely on vectors or vector systems to transport gene constructs to target cells. Methods for introducing nucleic acids into target cells and tissues include physical, biological and chemical methods. Physical methods for introducing polynucleotides, such as RNA, into target cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. RNA can be introduced into target cells using commercial methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0086] Chemical means for introducing polynucleotides into target cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0087] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Before the formation of closed structures, the lipid components undergo self-rearrangement to trap water and dissolved solutes between the lipid bilayers (Ghosh et al., (1991) Glycobiology 5: 505-10). However, compositions with structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may adopt micellar structures or simply exist as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0088] Biological methods for introducing polynucleotide of interest into host cell include the use of DNA and RNA vectors.Viral vectors, especially lentiviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells.Viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc.See, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362.

[0089] In certain embodiments, the present invention includes a nucleic acid vector encoding a CLCN5 protein. In certain embodiments, the nucleic acid vector is a lentiviral vector. Lentiviral vectors are useful for transducing a nucleotide payload into a target cell. Once inside the cell, the RNA genome of the vector is reverse transcribed into DNA and integrated into the genome of the target cell. Lentiviral vectors are part of a large group of retroviral vectors. A detailed list of lentiviruses can be found in (Coffin et al. (1997) "Retroviruses" Cold Spring Harbor Laboratory Press. pp 758-763).

[0090] Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Non-primate lentivirus group includes the prototype "slow virus" Visna / Maedi virus (VMV), as well as the related Caprine Arthritis Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV) and the more recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV). Lentiviruses are different from other members of the retrovirus family in that they have the ability to infect both dividing and non-dividing cells, which makes them attractive vectors for in vivo gene therapy (Lewis et al (1992) EMBO J 11(8):3053-3058) and Lewis and Emerman (1994) J Virol 68 (1):510-516).

[0091] The basic structure of the genomes of retroviruses and lentiviruses share many common features, such as 5' and 3' LTRs (between or within which is located a packaging signal that allows the genome to be packaged), a primer binding site, an integration site that allows integration into the host cell genome, and the gag, pol and env genes that encode the viral particle components.

[0092] In provirus, i.e. the nucleic acid molecule of the vector that is integrated into the target cell genome, the viral and payload genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for the integration and transcription of provirus. LTRs can also act as enhancer-promoter sequences, controlling the expression of viral and payload genes.

[0093] The LTRs themselves are identical sequences and can be divided into three elements called U3, R and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary greatly between different viruses.

[0094] To make lentiviral vectors replication-incompetent in target cells, most vectors have deletions or mutations in gag, pol and env genes that render them absent or non-functional. In certain embodiments, the lentiviral vectors of the present invention may contain one or more of these modifications that render the viral vector replication-defective.

[0095] In certain embodiments, the lentiviral vector of the present invention can be a self-inactivating lentiviral vector. Self-inactivating retroviral vectors contain deletions of transcription enhancers and / or promoters in the U3 and U5 regions of LTR. However, any promoter contained within the transducing DNA sequence between LTRs in such vectors still remains transcriptionally active. This strategy has been used to eliminate the effect of enhancers and promoters in viral LTRs on the transcription from genes placed inside. Such effects include increased transcription (Jolly et al (1983) Nucleic Acids Res. 11:1855-1872) or repression of transcription (Emerman and Temin (1984) Cell 39:449-467). This strategy can also be used to eliminate downstream transcription from 3'LTR to genomic DNA (Herman and Coffin (1987) Science 236:845-848). Such modifications are particularly useful in lentiviral vectors used in human gene therapy where activation of endogenous oncogenes should be avoided.

[0096] Regardless of the method used to introduce nucleic acid into cell, various assays can be carried out to confirm the presence of nucleic acid in cell.Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein to identify payload proteins that fall within the scope of the present invention.

[0097] Treatment methods In certain aspects, the nucleic acid vector described herein is a lentivirus vector.In certain aspects, the nucleic acid vector can be included in a pharmaceutical composition useful for treating Dent's disease in a subject in need thereof.The composition can include a pharmaceutical composition and can further include a pharmaceutically acceptable carrier.A therapeutically effective amount of the pharmaceutical composition comprising the nucleic acid vector can be administered.

[0098] In one aspect, the present invention includes a method for treating Dent's disease in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid vector encoding a CLCN5 protein, thereby treating the disease. In another aspect, the present invention includes a method for correcting a mutation in the CLCN5 gene in a cell, comprising contacting the cell with a nucleic acid vector encoding a functional CLCN5 protein. In certain embodiments of the above aspects, the nucleic acid vector is a lentiviral vector.

[0099] In certain embodiments, the lentiviral vectors and compositions of the present invention are delivered locally to target tissues, including various sites in the kidney. In certain embodiments, delivery of CLCN5 protein is most beneficial when targeting cells that depend on the expression of CLCN5 protein for their normal function. In the kidney, these cells include, but are not limited to, epithelial cells that line the proximal tubule and the thick ascending limb of Henle's loop and are in the interstitial cells of the collecting duct. In certain embodiments, local administration of the lentiviral vectors or compositions of the present invention to the kidney is achieved via retrograde ureteral injection. In this way, lentiviral particles directly contact the target tissue through the lumen of the tubule and duct. In certain embodiments, retrograde injection is followed by temporary or partial ligation of the ureter to prevent lentiviral particles from flowing out of the kidney tissue before they can contact the target cells.

[0100] Mouse model of Dent's disease Dent's disease is a genetic disease caused by deleterious mutations in genes including CLCN5. Thus, experimental models including genetically modified mice are useful tools for studying the biological aspects of the disease as well as for developing potential treatments for Dent's disease, including those disclosed herein. Thus, in another aspect, the present invention includes a mouse model for studying Dent's disease type 1, in which the CLCN5 gene of the mouse is disrupted by one or more mutations. A number of mutations can result in the inactivation or reduced activation of a particular gene by changing the structure of the resulting protein or suppressing the production of the protein all together. Such mutations include, but are not limited to, missense, frameshift and nonsense mutations. In certain embodiments, the mutation can be in the region that controls the post-transcriptional process of the mRNA encoded in the gene, including, but not limited to, splicing, among other processes. In certain embodiments, the mutation is a deletion that includes one or more exons of the CLCN5 gene. In certain embodiments, the deletion affects exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11 of the CLCN5 gene, or any combination thereof. Thus, in certain embodiments, the one or more CLCN5 mutations result in a non-functional CLCN5 protein.

[0101] In certain mouse models, this genetic mutation or change affects the animal's reproductive or fertility. In many cases, these effects are detrimental, since they produce few or no offspring with the desired genotype required for the model. One way to reduce or avoid these negative effects on reproductive and fertility is to outcross the experimental mouse with another strain, since the severity of fertility problems is often strain-specific. Thus, in certain embodiments of the present invention, breeding of experimental animals requires sires and dams of different strains. In one non-limiting example, the dam is heterozygous for CLCN5 mutation and the sire is wild type. This setup reproduces the X-linked inheritance commonly seen in Dent's disease. In certain embodiments, the sire is of FVB background, while the dam is of C57BL / 6 background. It is also contemplated that the sires and dams of the mouse models of the present invention may be of any number of different strains, including, but not limited to, BALB / C and derivatives, C3H and derivatives, DBA and derivatives, C57BL / 10 and derivatives, and other derivatives of the C57BL / 6 and FVB strains or any combination thereof. One of skill in the art will recognize the relative advantages of various laboratory mouse strains when selecting the two for use in the mouse models of the present invention.

[0102] Pharmaceutical Compositions The pharmaceutical composition of the present invention may comprise one or more pharma- ceutically or physiologically acceptable carriers, diluents, adjuvants or excipients in combination, as described herein.Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.The composition of the present invention is preferably formulated for intravenous administration.

[0103] The pharmaceutical composition of the present invention can be administered in a manner and mode or route of administration appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0104] The pharmaceutical compositions of the present invention may be administered in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, emulsions, etc. The pharmaceutical compositions of the present invention may be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by nasal drops, by implantation, by intracavity or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. In some embodiments, the compositions may be applied to the nose, throat, or bronchi, for example, by inhalation.

[0105] Optionally, the methods of the invention provide for administration of the compositions of the invention to a suitable animal model to determine the dosage of the composition, the concentration of components therein, and the timing of administration of the composition that induces tissue repair, reduces cell death, or induces another desired biological response. Such determinations do not require undue experimentation and are routine and can be ascertained without undue experimentation.

[0106] Biologically active agents can be conveniently provided to subjects as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH. The lentiviral vectors and agents of the present invention can be provided as liquid or viscous formulations. In some applications, liquid formulations are desirable, especially because they are convenient to administer by injection. Viscous compositions may be preferred when prolonged contact with tissue is desired. Such compositions are formulated within an appropriate viscosity range. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, such as water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0107] Sterile injectable solutions are prepared by suspending talampanel and / or perampanel, if desired, in the required amount of an appropriate solvent with various amounts of other ingredients. Such compositions may be in admixture with suitable carriers, diluents or excipients, such as sterile water, saline, glucose, dextrose, and the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and preparation desired. Suitable preparations may be prepared without undue experimentation, following guidance from standard textbooks, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition (1985), incorporated herein by reference.

[0108] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers.The inhibition of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.The extended absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.However, according to the present invention, any vehicle, diluent or additive used must be compatible with the cells or agents present in the conditioned medium.

[0109] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears.The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride, or other pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride is particularly preferred for buffers that contain sodium ions.

[0110] The viscosity of the composition can be maintained at a selected level if desired using a pharma- ceutically acceptable thickening agent such as methylcellulose.Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc.The selection of suitable carriers and other additives will depend on the exact route of administration and the nature of a particular dosage form, for example, a liquid dosage form (for example, whether the composition should be formulated into a solution, suspension, gel, or another liquid form, for example, a sustained release form or a liquid-filled form).Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert.

[0111] It is to be understood that the methods and compositions that may be useful in the present invention are not limited to the particular formulations shown in the examples.

[0112] The practice of the present invention employs, unless otherwise specified, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology that are well within the understanding of those skilled in the art. Such techniques are fully described in such publications as "Molecular Cloning: A Laboratory Manual" 4th Edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be taken into consideration in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed herein.

[0113] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0114] Experimental Example The present invention will be described in more detail by reference to the following experimental examples. Unless otherwise specified, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any variations that become evident as a result of the teachings provided herein.

[0115] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples, therefore, specifically point out illustrative aspects of the present invention, and are not to be construed as limiting the remainder of the disclosure in any way.

[0116] The materials and methods used in the following experimental examples will now be described.

[0117] Study Approval. Experiments were performed in accordance with the National Research Council Publication Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Wake Forest University Health Sciences (Animal Protocol number A19-053). Mice were housed in microisolator cages with a 12-h light / dark cycle and fed ad libitum. Mice were euthanized using carbon dioxide (CO2) overdose, which caused rapid loss of consciousness and subsequent death. Mice were exposed to CO2 without being removed from their home cages, ensuring that the animals were not stressed by being handled or moved to a new environment. CO2 flow rates were set to replace 10%-30% of the cage volume per minute. Once mice exhibited deep coma, they were subjected to cervical dislocation as a secondary method of euthanasia. After euthanasia, kidney tissues were collected for further analysis.

[0118] Constructs. The lentiviral vector plasmid pCSII-hCLCN5 was constructed to express a codon-optimized human CLCN5 cDNA under the control of the human EF1 alpha promoter. Plasmid pCSII-hCLCN5 was generated by replacing the XhoI-XbaI fragment of pCSII-EF-miRFP709-hCdt(1 / 100) (Addgene Plasmid #80007) with a synthetic, codon-optimized cDNA encoding the human CLCN5 protein (see Table 1 for cDNA and protein sequences). Gene synthesis was performed by GenScript Inc. and sequences were confirmed by Sanger sequencing. Plasmids pMD2.G (Addgene #12259), pMDLg / pRRE (Addgene #12259) and pRSV-Rev (Addgene #12253) were purchased from Addgene and have been previously described. ZsGreen-expressing and GFP-expressing lentiviral transfer plasmids pLVX-IRES-ZsGreen1 and CmiR0001-MR03 were purchased from Takara Bio and GeneCopoeia, Inc., respectively. Sequence information for the primers is listed in Supplementary Table 1.

[0119] Generation of CLCN5 null mice. CLCN5 null mutant mice were generated by CRISPR / Cas9-mediated knockout of the mouse Clcn5 gene. Mouse Clcn5 intron 2 TIFF2024520416000001.tif4128, Intron 5 TIFF2024520416000002.tif4128 and exon 12 Three single guide RNAs (sgRNAs) targeting TIFF2024520416000003.tif4128, respectively, were injected into fertilized mouse eggs together with Streptococcus pyogenes Cas9 (SpCas9) mRNA to generate targeted knockout offspring. F0 founder animals were identified by PCR followed by sequence analysis, and these were crossed with wild-type mice to generate F1 animals. Successful deletions would generate lines in which the genomic DNA region encoding 711 aa (95%) of the 746 aa CLCN5 protein was deleted. RNA microinjection into fertilized eggs was performed at Cyagen (Biotechnology Company, Santa Clara, California). Founder heterozygous mice on a C57 / BL6 background were then housed in a pathogen-free animal facility at the Wake Forest University School of Health Sciences. To avoid partial embryonic or perinatal lethality of mutant mice on a C57 / BL6 background, mice were crossed onto a 50% FVB and 50% C57 / BL6 background.

[0120] Genotyping of mutant mice. Tail or ear pieces were digested with proteinase K (400 μg / ml) in PCR buffer containing 0.45% NP40 at 55°C for 3 h or overnight. Proteinase K was inactivated at 95°C for 13 min. The cleared lysates were used directly for PCR. PCR primers CLCN5-KF2 TIFF2024520416000004.tif4128 and CLCN5-KR2 TIFF2024520416000005.tif4128 was used to amplify a product of approximately 1000 base pair (bp) band from the mutant allele. CLCN5-KF2 and CLCN5-W2 TIFF2024520416000006.tif4128 was used to amplify a 540 bp product from the wild type allele. PCR cycling included an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 60 s / kb, and a final extension step at 72°C for 5 min. Wild type, heterozygous and homozygous mutant mice show only the 540 bp band, both the 540 bp and 1000 bp bands, and only the 1000 bp band, respectively, in these two PCRs.

[0121] Isolation and culture of renal proximal tubule cells. Renal cortex was finely minced and incubated with collagenase (Worthington Biochemical, Freehold, NJ) and soybean trypsin inhibitor (GIBCO Laboratories, Grand Island, NY), both at a concentration of 0.5 mg / ml, for 30 min. After removing large undigested fragments by gravity, the suspension was mixed with an equal volume of 10% horse serum in Hank's Balanced Salt Solution and then centrifuged at 500 rpm for 7 min at room temperature. The pellet was washed once by centrifugation and then suspended in serum-free cell culture medium, a mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mix (1:1) containing 2 mM glutamine, 15 mM N 2 hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 500 U / ml penicillin, and 50 pg / ml streptomycin. The pelleted tissue fragments were resuspended in high glucose DMEM medium containing 10% FBS, 1% L-glutamine and 1% penicillin streptomycin supplement and incubated in tissue culture dishes at 37°C with 5% CO2 to allow epithelial cells to grow out of the tissue and adhere to the bottom of the dish. After two passages, the cells were dissociated by trypsinization and seeded at 8 x 104 cells / well in 24-well plates for LV transduction.

[0122] Production of lentiviral vectors. Lentiviral transfer plasmids pCSII-hCLCN5, CmiR0001-MR03 and pLVX-IRES-ZsGreen1 were used to produce lentiviral vectors expressing the respective transgenes in a third generation packaging system. Briefly, 13 million actively growing HEK293T cells in a 15 cm dish were replaced with 15 ml of Opti-MEM. The following DNAs were used for co-transfection: 12 μg lentiviral transfer plasmid DNA (pCSII-hCLCN5, CmiR0001-MR03 or pLVX-IRES-ZsGreen1), 14 μg pMDLg / pRRE, 6 μg pMD2.G and 4 μg pRSV-Rev. DNAs were mixed in 1 ml of Opti-MEM. In a separate tube, 108 μl of polyethyleneimine (1 mg / ml, PEI, Synchembio, Cat # SH-35421) was added to 1 ml of Opti-MEM. The DNA and PEI mixtures were then mixed and incubated at room temperature for 15 minutes. The DNA / PEI mixture was then added to the cells in Opti-MEM. 24 hours after transfection, the medium was replaced with 15 ml of Opti-MEM, and the lentiviral vectors were harvested at 48 and 72 hours after transfection. The combined supernatants were spun at 500 g for 10 minutes to remove cell debris. For in vivo delivery, the cleared supernatants were further processed as described below.

[0123] Concentration of lentiviral vectors. The supernatant containing the lentiviral vectors was first concentrated using a KR2i TFF System (KrosFlo® Research 2i Tangential Flow Filtration System) (Spectrum Lab, Cat. No. SYR2-U20) using the concentration-diafiltration-concentration mode. Briefly, 150-300 ml of the supernatant was first concentrated to about 50 ml, diafiltered with 1000 ml of PBS, and finally concentrated to about 8 ml. The hollow fiber filter module was made of modified polyethersulfone and had a molecular weight cutoff of 500 kDa. The flow rate and upper pressure limit were 80 ml / min and 8 psi for filter module D02-E500-05-N and 10 ml / min and 5 psi for filter module C02-E500-05-N.

[0124] To further increase vector concentration for in vivo delivery, 4 volumes of TFF concentrated vector were prepared per volume of 10% sucrose buffer (in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5 mM EDTA). Viral vectors were centrifuged at 10000g for 4 hours at 4°C and resuspended in approximately 0.5 ml of PBS. Vectors were aliquoted into 100 μl / tube and frozen at -80°C for future use.

[0125] Quantification of Lentiviral Vectors. Lentiviral vector concentrations were determined by p24 (capsid antigen)-based ELISA (Cell Biolabs, QuickTiter™ Lentivirus Titer Kit Catalog Number VPK-107). Concentrated vectors were diluted 200-fold for the assay. To assay unconcentrated samples, viral particles were precipitated according to the manufacturer's instructions, and no soluble p24 protein was detected.

[0126] Retrograde ureteral injection. Lentiviral vectors were delivered to the kidney by retrograde ureteral injection as previously reported. Mice were anesthetized with 3% isoflurane inhalation, and the left kidney was exposed through a 2 cm flank incision and gently dissected from the surrounding fat. A non-invasive vascular clip (S&T Vascular Clamps Cat# 00400-03, Fine Science Tool, Heidelberg, Germany) was placed on the ureter below the injection site to prevent leakage into the bladder. Lentiviral particles were injected into the ureter just below the ureteropelvic junction using a 30-gauge 1 / 2 needle connected to a 1 ml syringe. The total volume of the viral solution did not exceed 100 μl. The concentration of the viral vector was 2–4 ng / μl. After 5–15 min, the clamps were removed and the surgical site was closed by joining the two skins together with absorbable 5-0 Vicryl sutures. If bilateral injections were performed, the same procedure was repeated for the right kidney after closing the left incision. Immediately after surgery and before recovery, mice were given three doses of 5–10 mg / kg carprofen (one dose per 24 h). Along with the first carprofen injection, buprenorphine SR (0.5–1.0 mg / kg) was also given by subcutaneous injection. After recovery from surgery, mice were housed singly. Singly housing was found to prevent cage-mate-related wound injury.

[0127] Urine collection. Mice were housed in Hatteras Instruments Model MMC100 Metabolic Mouse Cages (Hatteras Instruments Inc, 105 Southbank Dr, Cary, North Carolina) for 24 hours for urine collection. Urine samples were briefly spun at 1000g for 5 minutes to remove potential particles. Urine volume was measured with a 200 μl pipette.

[0128] Urine biochemistry. Urinary calcium concentration was determined with a Calcium Assay Kit (colorimetric) (ab102505, AbCam). Urine samples from wild-type and CLCN5 LV-treated mice were diluted 3.6-fold and urine samples from untreated mutant mice were diluted 10-fold with water prior to assay. Total calcium excretion was calculated by multiplying calcium concentration by the respective urine volume collected during a 24-hour period. Urinary total protein concentration was determined with a Pierce™ BCA Protein Assay kit (Cat#23225). All urine samples were diluted 10-fold with water prior to assay. Total urinary protein excretion was calculated by multiplying urine protein concentration by the respective urine volume collected during a 24-hour period. Urinary creatinine was assayed with a Mouse Creatinine Assay Kit (Crystal Chem Inc., #80350). Urine samples were diluted 10-fold with saline prior to assay. All measurements were performed according to the kit instructions.

[0129] SDS-PAGE and Western blotting analysis. Mouse kidney tissues were lysed in RIPA buffer containing protease inhibitors (0.5 mM PMSF and 1× Complete Protease Inhibitor Cocktail, Roche Diagnostics Corporation, Indianapolis, IN, USA) and phosphatase inhibitors (50 mM NaF, 1.5 mM Na3VO3), and the lysates were mixed with Laemmli buffer for SDS-PAGE for Western blotting analysis. Cultured cells and urine samples were directly lysed in 1× Laemmli buffer containing protease inhibitors and phosphatase inhibitors. Anti-β-actin antibody was from Sigma (A5441, 1:5000; St Louis, MO, USA), CLCN5 rabbit polyclonal antibody was from GeneTex (GTX53963, 1:500, Irvine, CA, USA), CC16 rabbit polyclonal antibody was from BioVendor (RD181022220-01, 1:500, Asheville, NC, USA), albumin goat polyclonal antibody was from Bethyl Laboratories (A80-129A, 1:1000, TX, USA), DBP rabbit polyclonal antibody was from Proteintech (16922-1-AP, 1:1000, IL, USA), and megalin rabbit polyclonal antibody (ab76969, AbCam) was from AbCam. HRP-conjugated anti-mouse IgG (H+L) (ThermoFisher Scientific, Cat No. 31430, 1:5000) and anti-rabbit IgG (H+L) (Cat No. 31460, 1:5000) secondary antibodies were used in Western blotting. Protein signals were visualized under the LAS-3000 system (Fujifilm) using chemiluminescence reagents (ThermoFisher).

[0130] Immunofluorescence analysis. Renal tissues were fixed overnight at 4°C in 4% paraformaldehyde / PBS. Part of the tissue was embedded in OCT for frozen sectioning, and part was dehydrated and embedded in paraffin. 5-8 μm paraffin sections were prepared for histological and immunofluorescence analysis. For immunofluorescence staining, deparaffinized and rehydrated sections were incubated with primary antibodies (1:200 for CLCN5 and megalin antibodies) following blocking, and then incubated in Alexa fluor 488 or CF-594-conjugated secondary antibodies. Sections were mounted in mounting medium containing DAPI (Vector Laboratories). Images were acquired with an Axio M1 microscope (Carl Zeiss, Thornwood, NY, USA) equipped with an AxioCam MRc digital camera. The different images were combined into one file in Adobe Photoshop with necessary resizing, rotation, and cropping. Fluorescence intensity was analyzed by NIH ImageJ (1.49v).

[0131] Detection of vector DNA. Each kidney was cut into 12 pieces, one of which was used for genomic DNA isolation using the DNeasy Blood & Tissue Kit (Qiagen). To detect lentiviral vector integration, the Psi sequence from the lentiviral vector was detected by qPCR using Psi-F and Psi-R primers and SYBR Green Master Mix (Thermo Fisher Scientific). Mouse Gapdh was used as an internal control, and TaqMan Universal PCR Master Mix and Gapdh Taqman probe (Thermo Fisher Scientific) were used for qPCR detection.

[0132] RNA isolation and RT-qPCR analysis. Total RNA was isolated from tissues and cultured cells using the miRNeasy Mini Kit (QIAGEN Cat No. 217004). RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN). RT-qPCR was performed on a QuantStudio3TM or ABI 7500 instrument using the primers listed in Supplementary Table 1.

[0133] Statistical analysis. Statistical evaluation was performed on urinary parameters and immunostaining data using GraphPad Prism (V5) software. Data are presented as mean ± standard error of the mean (SEM). For data analysis involving two groups, statistical differences between groups were calculated using two-tailed t-test. For data analysis involving more than two groups, one-way analysis of variance (ANOVA) was performed for all parameters. When ANOVA revealed significance, Tukey's post-hoc test was performed for data analysis. For data analysis involving more than one factor, two-way analysis of variance (ANOVA) was performed for all parameters. When ANOVA revealed significance, Bonferroni post-hoc test was performed for data analysis. Significance was set at *p<0.05, **p<0.01 and ***p<0.0001.

[0134] Table 1. Sequences of vectors, polynucleotides, proteins and primers used in the present invention TIFF2024520416000007.tif74148TIFF2024520416000008.tif221148TIFF2024520416000009.tif221148TIFF2024520416000010.tif221148 TIFF2024520416000011.tif221148TIFF2024520416000012.tif221148TIFF2024520416000013.tif222148TIFF2024520416000014.tif43148

[0135] Example 1: CLCN5 null mice exhibit a typical Dent's disease type 1 (DD1) phenotype Dent's disease is caused by the inability of kidney cells to reabsorb nutrients, water and other substances filtered from the bloodstream. The large amount of protein and calcium in the kidney filtrate damages kidney cells, ultimately causing the symptoms observed. The study of this disclosure aimed to develop a useful animal model of Dent's disease to aid in the development of gene therapy for Dent's disease. The ultimate goal is to correct the mutated gene in a minimum percentage of patient kidney cells so that they can reabsorb enough substances from the kidney filtrate to prevent damage to kidney cells and restore or maintain normal kidney function.

[0136] At least two genes (both X-linked and maternally inherited) can cause the disease, but abnormalities in one of the genes (CLCN5 gene) are responsible for 60% of patients (Dent disease type 1). A wide range of mutations have been identified in CLCN5, with missense, frameshift and nonsense mutations accounting for the majority (see Figure 1). Therefore, in this disclosure, we develop a gene therapy strategy specific for Dent disease type 1. Currently, there are only supportive treatments for Dent disease, none of which target the genetic causes of the disease.

[0137] In order to develop a treatment for Dent's disease, we first conducted research to create a suitable mouse model of the disease. Here, we used CRISPR / Cas9 technology to target most of the coding region of the CLCN5 gene for deletion (Figure 2A). Three guide RNAs were designed to target mouse CLCN5 gene intron 2, intron 4 and exon 12, respectively, to delete 95% of the protein coding region and completely disrupt gene function (Figure 16A). Three single guide RNAs (sgRNAs) and Cas9 mRNA were injected into fertilized mouse eggs to delete the CLCN5 gene. Three heterozygous founder female mice were obtained, all of which had a 26 kilobp deletion in the Clcn5 gene that deleted 95% of the CLCN5 coding region (Figure 21). There were no other known coding or non-coding genes within 80 kilobp around the deleted region. Therefore, we hypothesized that the deletion of the CLCN5 gene is unlikely to affect other genes. Offspring from one carrier female (no. 34) were used in subsequent studies.

[0138] Considering that the mice were generated by CRISPR / Cas9-mediated gene mutation, we analyzed the possible off-targets of the three sgRNAs used (we used three sgRNAs instead of two to increase the chance of deleting the whole gene). All predicted off-targets had at least 3nt mismatches to the sgRNA. Only one of the predicted off-targets (for sgRNA 2) hit an exon of a protein-coding gene (Itgb6). DNA of this region was amplified from male mutant mice and sequenced. No mutations or heterozygosity were observed (Figure 30). Eighteen predicted off-targets were in introns and 23 were in intergenic regions. We amplified the regions of all four predicted off-targets on the X chromosome from male mutant mice, but could not detect any mutations or deletions (Figure 30). Since the off-targets on the X chromosome are associated with CLCN5 deletion and male mice only have one copy of the X chromosome, successful amplification of this region also excluded the possibility of a large deletion. For autosomal off-targets, we sequenced the regions of all five off-targets with 3nt mismatches to the sgRNA and two off-targets with 4nt mismatches to the sgRNA (at least three off-targets per sgRNA were analyzed) and could not detect mutations or heterozygosity in any of these regions in mutant mice (Figure 30). Collectively, the data indicated that the likelihood of other genes being unintentionally mutated was low.

[0139] RT-PCR (Figure 16B) and Western blotting (Figure 16C) confirmed the loss of CLCN5 expression in the mutant mice. Mutant animals resulting from these studies were sequenced, which confirmed the excision of exons 3-11 (Figure 2B), thus generating a model completely lacking CLCN5 function. Detection of both CLCN5 mRNA and CLCN5 protein then confirmed that the mutant mice lacked expression of the CLCN5 gene product (Figure 4). Urine from wild-type and mutant mice was collected and the total urine volume excreted over a 24-hour period, total urinary protein and calcium levels were measured. Female and male mutant mice exhibited diuresis, hypercalciuria and proteinuria (Figures 25A-25B). Wild-type female mice exhibited higher urinary calcium levels than wild-type male mice, which is consistent with previous findings. Interestingly, heterozygous female mice also displayed a DD1-like phenotype that appeared less severe than homozygous mutant female mice, suggesting haploinsufficiency and consistent with reports that some human female heterozygous carriers display milder DD1 symptoms. The phenotype observed in these null mutant mice (6-7-fold increase in urinary protein and urinary calcium) was much more severe than previously reported, likely due to deletion of a large portion of the Clcn5 coding sequence. Urinary creatinine concentrations in mutant mice were similar to those in wild-type mice, suggesting that creatinine filtration in mutant mice was not significantly affected at the time of analysis.

[0140] Unexpectedly, reproductive failure led to obtaining an insufficient number of diseased male mice. The observed ratio of diseased to normal male mice (Figure 3A) suggested embryonic lethality. This problem had not been observed in previous studies or in humans. To restore the expected Mendelian ratio in the offspring, we developed a breeding strategy to separate female carriers and normal males into a different breed (a mix of FVB and C57BL / 6) (Figure 3B).

[0141] The mutant mice were then examined to see if they displayed a phenotype similar to that observed in DD1 patients. Urine from normal and mutant mice and measured diuresis, total urinary protein and calcium levels (Table 2). Male and female mutant mice urinated more frequently and excreted more urinary protein and calcium than normal mice (Table 3). Normal female mice showed higher urinary calcium levels than normal male mice, which is consistent with previous findings. Interestingly, heterozygous female mice also showed a less severe DD1-like phenotype than homozygous mutant female mice, suggesting haploinsufficiency and consistent with reports that some human female heterozygous carriers display mild DD1 symptoms. The phenotype observed in these null mutant mice (two-fold increase in urinary protein and six-fold increase in urinary calcium) was much more severe than previously reported, likely due to the deletion of a large portion of the CLCN5 coding sequence. Urinary creatinine concentrations in mutant mice were similar to those in wild-type mice, suggesting that creatinine filtration in mutant mice was not significantly affected at the time of study.

[0142] Consistent with the increased total urinary protein content (BCA assay) in mutant mice, SDS-PAGE analysis of urinary proteins confirmed the increased protein content in mutant urine, with a very strong 61 kDa unidentified protein in the urine samples of mutant mice, but barely visible in the urine samples of wild-type mice (Figure 5 and Figure 16D). The visibility of this strong band on SDS-PAGE was a reliable predictor of other DD1 phenotypes. Western blotting further confirmed the increase in urinary albumin, vitamin D binding protein (DBP) and club cell secretory protein (CC16, also called CC10) in samples from mutant mice (Figure 6 and Figure 16E). We loaded equal volumes of urine samples for SDS-PAGE and Western blotting experiments. Considering the increased urine volume in mutant mice, the degree of increase in urinary protein was more dramatic than seen in the Western blotting analysis. The data demonstrated that we successfully knocked out the mouse CLCN5 gene and that the mutant mice exhibited a more severe DD1 phenotype than observed in published models.

[0143] Table 2. Urinary protein and calcium values ​​in control and mutant mice TIFF2024520416000015.tif43148**, p<0.01; ***, p<0.0001. Each group contained 10 mice.

[0144] Table 3. Urine analysis of control and mutant mice TIFF2024520416000016.tif48148*, **, and *** indicate p<0.05, p<0.01, and p<0.0001 when the indicated groups were compared to wild-type mice by two-tailed unpaired t-test (males) or one-way ANOVA followed by Tukey's multiple comparison test (females).

[0145] Table 4. Urine analysis of mutant mice with both kidneys treated TIFF2024520416000017.tif52148

[0146] Example 2: Development of gene therapy for type I Dent disease Efforts to develop a strategy to correct the mutation that causes Dent's disease were focused on using lentiviral vectors to deliver a functional copy of the CLCN5 gene to cells in the kidney. Lentiviral vectors have been approved by the FDA as vehicles to deliver functional genes to human cells for gene therapy. The CLCN5-expressing lentiviral vector of the present invention was designed to express the same final protein product but with slight sequence differences from wild-type CLCN5 mRNA so that the virally delivered form of the mRNA could be distinguished from the original endogenous form (illustrated in FIG. 7 and also see Table 1). This was achieved by codon optimization of the CLCN5 mRNA expressed by the transgene. The transfer plasmid was a third generation lentiviral expression vector containing a codon-optimized human CLCN5 cDNA after the human EF1 alpha promoter (FIG. 17A, Table 1). Using a ubiquitously active promoter, we investigated whether supplementing the kidney with functional CLCN5 cDNA could ameliorate DD1 symptoms. To increase target gene expression, we included the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) after the CLCN5 cDNA. The CLCN5-expressing lentiviral vector was produced using a third-generation packaging system, and exogenous CLCN5 mRNA expression was successfully detected from HEK293T cells transduced with the lentiviral vector (Figure 17B).

[0147] In vitro studies on human 293T cells and primary renal cells isolated from normal and CLCN5 mutant mice transduced with CLCN5 vector or GFP-bearing control vector demonstrated that the transduced cells expressed robust levels of CLCN5 protein (Figure 8). Expression of the vector was then examined in renal proximal tubule cells isolated from wild-type and CLCN5 knockout mice transduced with CLCN5 lentiviral vector. CLCN5 protein was detected in CLCN5 LV-transduced mutant cells, but not in GFP-LV-transduced mutant cells (Figure 17C).

[0148] The eventual clinical use of lentiviral CLCN5 constructs will require that viral particles be directed to cells in the kidney that most require functional CLCN5 protein, particularly those in the proximal tubule and the thick ascending limb of the loop of Henle, both sites of calcium transport. Thus, studies were performed to deliver lentiviral vectors directly to kidney tissue by ureteral ligation followed by retrograde ureteral injection. Transiently tying off the ureter prevents lentiviral particles from spilling out before they have a chance to transfect kidney cells, while at the same time, injection of cells into the ureter allows the viral particles to reach the target tissue (Figure 9A). To confirm successful transduction of kidney cells using this method, mice were injected with GFP-expressing lentivirus using this technique. This method was then used to deliver 280 ng of p24 CLCN5 LV to the kidneys of mutant mice. One week later, kidney tissue was harvested and assessed for GFP expression by fluorescence microscopy, which demonstrated readily visualized GFP+ cells (Figure 9B). Western blotting analysis of proteins extracted from kidney tissues revealed that CLCN5 protein was detectable in the kidneys of injected but not uninjected mutant mice (Figure S17D). These data demonstrated that LV vectors could be delivered to the kidney and CLCN5 expression could be obtained from the delivered lentiviral vector.

[0149] In a subsequent study, we investigated the delivery of GFP-LV to mouse renal tubules using retrograde ureteral injection. Two weeks after delivery of 100 μl of GFP-LV vector (approximately 250 ng p24) to each kidney, immunofluorescence detected strong GFP expression in more than 70% of the tubular structures in all four injected male mice (one 6-month-old wild type and three 17-month-old mutants) (Figure 26A). No GFP expression was observed in the glomeruli, suggesting that this delivery method is more suitable for delivery to the renal tubules than to the glomeruli. We collected the kidneys, bladder, liver, heart, skeletal muscle, spleen and testes of mice that had received GFP-LV retrograde ureteral injection, extracted genomic DNA and detected lentiviral vector DNA. Relatively low levels of vector DNA were detected in the bladder and 10% in the kidney. 5 2-fold higher levels of vector DNA were detected in the ureteral injection compared with the control group, and vector DNA was undetectable in all other organs (Figure 26B). These data indicated that retrograde ureteral injection is efficient for local tubular delivery and has a low chance of delivering vector to other organs (except for the bladder and possibly other tissues of the urinary tract).

[0150] We then used this method to deliver 280ng of p24 CLCN5 LV to the kidneys of male mutant mice. Western blotting analysis of proteins extracted from kidney tissues showed that CLCN5 protein was detected in the injected kidneys but not in the uninjected kidneys 2 weeks after vector delivery (Figure 17D). Immunofluorescence analysis was performed to examine the cell types expressing transgenic CLCN5. In the kidneys of wild-type mice, CLCN5 was highly expressed in the proximal tubule epithelium (Figure 27A), but weakly expressed in the glomerulus (inset of Figure 27A, marked with *). Without CLCN5 LV vector delivery, CLCN5 expression was not detected in the renal tubules of mutant mice (Figure 27B). Two weeks after CLCN5 LV delivery, CLCN5 was detected in the renal tubules of mutant mice (Figure 27C). The strongest CLCN5 signal was detected in the apical region of tubular cells in both wild-type and CLCN5 LV-injected mutant mice. The apical localization of exogenous CLCN5 protein indicated that LV-expressed CLCN5 protein was correctly transported. The data showed that retrograde ureteral injection can deliver LV vectors to the kidney and result in CLCN5 expression from the delivered lentiviral vector.

[0151] To investigate the cell types expressing transgenic CLCN5, we performed immunofluorescence analysis 2 weeks after vector delivery. In wild-type mice, CLCN5 was highly expressed in the proximal tubular epithelium (upper image in FIG. 17E, marked with *), but not in the glomerulus (marked with #). Without vector delivery, CLCN5 expression was not detected in mutant mice (middle image in FIG. 17E). With CLCN5 LV delivery, CLCN5 was detected in essentially all cells in the kidneys of mutant animals, including tubular structures (marked with *) and glomeruli (marked with #) (lower image in FIG. 17E). Overall, the level of CLCN5 expression in LV-delivered mutant animals was lower than that in wild-type mice. LV vector-mediated CLCN5 expression was consistent with the ubiquitous EF1 alpha promoter used to control CLCN5 expression.

[0152] Example 3: Delivery of human CLCN5 lentiviral vector to the kidneys of mutant mice ameliorated the DD1 phenotype We then carried out a study to assess whether intrarenal delivery of CLCN5-carrying lentiviral vectors could correct CLCN5 expression in CLCN5 knockout mice (Figure 10). It has been reported that CLCN5 deficiency causes a decrease in proteins involved in endocytosis, such as megalin and cubilin. We examined the expression of megalin and confirmed that it was decreased in the kidneys of mutant mice (Figure 18A). We then delivered 280ng of p24 CLCN5 LV to the kidneys of mutant mice, and in addition to restoring the expression of CLCN5 (Figure 18E), we also observed that the expression of megalin was slightly increased (Figures 18A, 18B), but the expression of megalin was still lower than that in wild-type mice.

[0153] The study then determined whether delivering CLCN5 LV to the kidneys of mutant mice could improve the phenotype. Gene therapy experiments were then performed on three groups of male mutant mice: Group 1 received an injection of 280 ng of p24 ZsGreen LV in each kidney to serve as a negative control (5 mice); Group 2 received an injection of 280 ng of p24 CLCN5 LV in the left kidney (10 mice); Group 3 received an injection of 280 ng of p24 CLCN5 LV in each kidney (10 mice). One month after treatment, the three groups were examined for diuresis, urinary protein, and urinary calcium levels. The DD1 phenotype was not improved in mice injected with ZsGreen LV, but was greatly improved in CLCN5 LV-treated mice, regardless of whether one or both kidneys were treated (Table 4). After CLCN5 LV treatment, diuresis and urinary calcium levels returned to normal levels (see Table 2 for normal values ​​in male mice). Urinary protein excretion after treatment was reduced 3-4 fold compared to before treatment, but was still higher than normal.

[0154] Consistent with the reduction in total urinary protein content after gene delivery in the BCA assay, the intensity of the 61 kDa and <20 kDa bands in SDS-PAGE analysis was greatly reduced in the urine of mice that received CLCN5 LV injections, but not ZsGreen LV injections (Figure 18C, Figure 23). After delivery of CLCN5 LV to the left kidney (Figure 18B) or both kidneys (Figure 18C), Western blotting confirmed the reduction in urinary albumin, vitamin D binding protein (DBP) and club cell secretory protein (CC16, also called CC10). Equal volumes of urine samples were loaded for SDS-PAGE and Western blotting experiments. Considering the greatly reduced urine volume after CLCN5 gene therapy, the degree of reduction in urinary protein was even more dramatic. The age of CLCN5 LV-treated mice varied from 25 to 200 days, and all treated mice showed similar degrees of improvement. Thus, the timing of CLCN5 gene therapy appeared to have little effect on the therapeutic effect.

[0155] Example 4: Therapeutic effects lasted for up to 4 months after gene therapy Urine produced by the animals was then monitored periodically for up to 4 months until the effect of the lentiviral vector disappeared. Renal tissue was also harvested from selected animals at various time points for histological analysis of CLCN5 expression. Results demonstrated that even when only one kidney was treated with the lentiviral vector (280 ng p24), gene therapy significantly reduced urinary protein secretion by mutant mice as assessed by SDS-PAGE (Figure 11) and Western blots of albumin and vitamin D binding protein (Figure 12). Similarly, Western blotting of CCL16 secretion also showed a dramatic decrease in treated mutant animals (Figure 13). The therapeutic effect was found to follow a time course, being detectable after 1 and 2 months of treatment and disappearing only after 4 months of treatment (Figure 14 and Table 5). In mutant mice with one kidney treated, urinary protein and urinary calcium levels returned to pretreatment values ​​4 months after gene delivery, and diuresis was also elevated after 1 month of treatment (Figure 19A). In mutants with both kidneys treated, urinary protein excretion returned to pretreatment levels after 4 months of treatment, whereas diuresis and calcium excretion were still lower than pretreatment levels after 4 months of treatment (Figure 19A), but returned to pretreatment levels after 6 months of treatment. The biochemical assay data were supported by SDS-PAGE and Western blotting analysis of urinary proteins after 4 months of gene therapy (Figure 19B, Figure 24). Considering a typical mouse survival time of 3 years, the 2 months would correspond to roughly 50 months of human survival time.

[0156] In a related study, CLCN5 LV was also delivered to the left kidney of five mutant mice aged 62-162 days. In all treated mice, a sharp decrease in urinary protein and urinary calcium excretion was observed one month after CLCN5 gene therapy (Figures 31A-31C). This decrease was not caused by aging of the mice, as aging did not cause a significant decrease in these parameters (Figures 32A-32C). Another experiment was performed in which both kidneys of mutant mice aged 53-156 days were treated with CLCN5 LV, and for each CLCN5 LV-treated mouse, both kidneys of an age-matched mutant mouse were treated with ZsGreen LV. After 1, 2 and 3 months of treatment, each CLCN5 LV-treated mouse showed greatly improved diuresis (Figure 29A, Figure 33), calciuria (Figure 29B) and proteinuria (Figure 29C), with levels close to those of wild-type mice (dashed line). In contrast, ZsGreen LV-treated mice did not show improvement in these parameters. Again, the reduction in urinary protein one month after CLCN5 LV treatment was confirmed by SDS-PAGE (Figure 18C) and Western blotting (Figure 18E) analysis. Four months after both kidneys were treated with CLCN5 LV, diuresis and proteinuria returned to pretreatment (Figures 29A, 29C), but calciuria was still improved compared to pretreatment levels and ZsGreen LV-treated mice (Figure 29B). In addition, we treated both kidneys of five mutant mice aged 81-196 days with CLCN5 LV, and again, all of these mice responded to treatment (Figures 34A-34C). In these mice, urinary calciuria still improved after four months of treatment, but returned to pretreatment levels after six months of treatment (Figure 34B). These data indicated that the timing of CLCN5 gene therapy appeared to have little effect on the therapeutic efficacy. Consistent with the biochemical assays, SDS-PAGE (FIG. 24) and Western blotting (FIG. 19B) analyses of urinary proteins also revealed that urinary protein levels were reduced after 1, 2, and 3 months of treatment, but returned to pretreatment levels after 4 months of treatment.

[0157] Table 5. Urinary protein and calcium levels in mutant mice before and after gene therapy of one kidney. TIFF2024520416000018.tif33148*** indicates p<0.0001 compared to pre-treatment values. Each group contained 10 mice.

[0158] We then performed a study in which both kidneys of mutant mice were treated with a single injection of CLCN5 lentiviral vector (280ng p24). Untreated mutant mice and mutant mice treated with GFP-expressing lentiviral vector were used as controls. One month after treatment, urine volume and urinary protein levels were found to have almost returned to normal levels, and urinary calcium levels were 4-fold lower than those of untreated mutant mice, but still higher than those of normal mice (Figure 15).

[0159] Example 5: Immune rejection underlies loss of therapeutic efficacy Without wishing to be bound by theory, there may be several possible explanations for the loss of therapeutic effect after 4 months of treatment: 1) promoter silencing; 2) natural epithelial cell senescence and replacement; and 3) immune rejection of CLCN5-expressing cells. To find the most likely mechanism, a series of experiments were then carried out. Five months after receiving the first dose of CLCN5 LV in the left kidney, if the therapeutic effect of the first dose was lost, then a second dose of LV was delivered to the untreated right kidney of mutant mice (Figure 20A). If the loss of therapeutic effect was caused by promoter silencing or natural senescence of treated cells, we should observe the therapeutic effect after receiving the second dose. If it was caused by immune rejection, no therapeutic effect should be observed after the second dose.

[0160] As a positive control, naive mutants were treated similarly to validate the LV and delivery procedure (Figure 20, Animals No. 6 and No. 7). Urine samples were collected 15 days after vector delivery and a clear reduction in urinary protein was observed after treatment in naive mice (Figure 20B, Mouse No. 6), demonstrating the success of the procedure and the functionality of the vector. However, no reduction in urinary protein was observed in any of the five pre-treated mice (Animal Nos. 1-5), although these mice showed a clear reduction in urinary protein after the first dose of CLCN5 LV (Figure 20B). Diuresis, urinary protein and calcium excretion were improved only after the first dose, but not after the second dose (Figure 20C). These data suggested immune rejection as the most likely major mechanism of action.

[0161] We also examined LV DNA integration, human CLCN5 mRNA expression, and CLCN5 protein expression in the injected kidneys. LV DNA (Psi signal) (Figure 20D), human CLCN5 mRNA (Figure 20E), and CLCN5 protein (Figure 20F) were detectable in the kidneys of naive mice (animals no. 6 and 7), but were greatly reduced or undetectable in the kidneys of pretreated mice (animals no. 3, 4, and 5). In contrast to the lack of CLCN5 expression after delivery of a second CLCN5 LV injection to mice pretreated with CLCN5 LV, delivery of GFP LV to mice pretreated with CLCN5 LV resulted in robust GFP expression (Figure 35, CLCN5-LV, GFP-LV, no. 2-4). These data were consistent with the observation of a gene therapy effect 1 month after the first dose, but not after the second dose, further supporting the immune rejection response.

[0162] Example 6: Selected discussion In summary, without wishing to be bound by theory, these results demonstrate that it is possible to generate a mouse model of Dent's disease of the present invention in which CLCN5 expression is abolished, resulting in phenotypic and functional effects that reflect the clinical symptoms of Dent's disease in human patients. Notably, the Dent's disease mouse model of the present invention demonstrates a much more pronounced phenotype than the current Dent's disease mouse model generated through homologous recombination. One unexpected phenotype was partial embryonic or perinatal lethality of mutant mice on a C57 / BL6 background. This observation suggests that the CLCN5 gene may function during early development, which is consistent with the observation of CLCN5 expression during embryonic development and in organs other than the kidney. In addition, the mutant mice showed more severe proteinuria and hypercalciuria compared to published models. There are no other predicted genes (including non-coding genes) within 40 kilobp surrounding the deleted region. Thus, the observed phenotype was the result of deleting 95% of the CLCN5 coding region, which precluded the possibility of expressing a partially functional CLCN5 protein, and these null mutants may therefore be useful to study the physiological consequences of a complete lack of the CLCN5 protein.

[0163] The data disclosed herein show that gene replacement therapy can be an effective treatment option for DD1. CLCN5 LV vector was delivered to 22 mutant mice (10 mice with one kidney treated and 12 mice with both kidneys treated), and 100% of treated mice showed significant improvement in all of the parameters examined: diuresis, proteinuria, and hypercalciuria. After treatment, diuresis and urinary calcium levels were restored to normal, whereas urinary protein levels were reduced to 20% of pretreatment values, but were still 80% higher than normal. Thus, gene therapy is highly effective in improving the symptoms of DD1. Another interesting observation is that 25-day-old and 200-day (6.5 months)-old mice responded equally to the treatment, indicating that the timing of gene therapy is not critical in this disease. This observation is meaningful in clinical applications, since it is not only young patients who can benefit from gene therapy.

[0164] CLCN5 is also expressed in the intestinal epithelium, and one study raised the potential role of intestinal calcium absorption in the hypercalciuria of CLCN5-deficient mice. We delivered CLCN5 LVs to the kidney by retrograde ureteral injection and completely restored urinary calcium levels in mutant mice. These data suggest that CLCN5 expressed in the kidney plays a major role in calcium maintenance.

[0165] Frameshift and nonsense mutations account for 29% and 17.5% of all DD1-induced mutations, and these mutations are likely to result in the expression of truncated CLCN5 proteins or the total absence of protein, as seen in the model mice of the present disclosure. These data suggest that gene replacement therapy is most likely to be beneficial for these patients. Approximately 33% of DD1-induced mutations are missense mutations, which express unstable, rearranged or dysfunctional proteins. Gene therapy may be beneficial for some subjects expressing unstable or rearranged CLCN5 proteins. It remains to be seen to what extent gene therapy will be beneficial for subjects expressing dysfunctional CLCN5, since CLCN5 most likely forms homodimers, and endogenous dysfunctional CLCN5 proteins may interfere with the function of exogenous CLCN5 proteins.

[0166] In the study disclosed herein, the effect of gene therapy lasted up to 4 months. Consistent with the observation that gene therapy completely normalized urinary calcium levels but not urinary protein levels, the beneficial effect on hypercalciuria lasted longer than on proteinuria. Immune response appears to be the main mechanism underlying the loss of gene therapy effect, which was supported by the lack of therapeutic effect after the second dose of LV was delivered to pretreated mice. Attenuated gene therapy effect was first observed 2 months after gene delivery.

[0167] Immune responses against the transgene product have been observed previously. Because mutant mice do not express CLCN5 at all, constitutively expressed human CLCN5 protein expressed from the LV vector is expected to induce adaptive immune responses. It remains to be seen whether relatively mild immune responses will be observed in subjects expressing unstable or dysfunctional CLCN5 proteins.

[0168] The gradual loss of therapeutic efficacy due to host immune response suggests the importance of suppressing the host immune response to achieve long-term gene therapy efficacy. There are several strategies that help minimize the immune response. One is to use tissue-specific promoters to avoid transgene expression in dendritic cells (DCs), which are mediators of adaptive immune responses. In this study, we used the EF1 alpha promoter, which is active in essentially all cells, for proof-of-concept. Because the proximal tubule is the main site of reabsorption, the use of tubule proximal cell-specific promoters, such as those against Npt2a or Sgtl2, may help reduce the immune response.

[0169] List of Aspects The following list of aspects is provided, the numbering of which should not be construed as indicating any level of importance. Aspect 1 provides: A method for treating Dent's disease in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid vector encoding a CLCN5 protein, thereby treating the disease. Aspect 2 provides: The method of embodiment 1, wherein the nucleic acid vector is a lentiviral vector. Aspect 3 provides: The method of embodiment 1, wherein said nucleic acid vector is operably linked to a promoter driving expression of a CLCN5 protein. Aspect 4 provides the following: The method of embodiment 3, wherein the promoter is a constitutive promoter. Aspect 5 provides the following: The method of embodiment 4, wherein the promoter is an EF-1α promoter. Aspect 6 provides the following: The method of embodiment 3, wherein the promoter is a tissue-specific promoter. Aspect 7 provides the following: The method of embodiment 6, wherein the tissue-specific promoter is specific to renal tubular proximal cells. Aspect 8 provides the following: The method of embodiment 7, wherein the tissue specific promoter is selected from the group consisting of Npt2a and Sgt12. Aspect 9 provides the following: The method of embodiment 2, wherein the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1. Aspect 10 provides the following: The method of embodiment 1, wherein said administering is delivered locally to the kidney. Aspect 11 provides the following: The method of embodiment 10, wherein the localized renal administration is delivered by retrograde ureteral injection. Aspect 12 provides the following: A method for correcting a mutation in a CLCN5 gene in a cell, the method comprising contacting the cell with a nucleic acid vector encoding a functional CLCN5 protein. Aspect 13 provides the following: The method of embodiment 12, wherein the nucleic acid vector is a lentiviral vector. Aspect 14 provides the following: The method of embodiment 12, wherein said nucleic acid vector is operably linked to a promoter driving expression of a CLCN5 protein. Aspect 15 provides the following: The method of embodiment 14, wherein the promoter is a constitutive promoter. Aspect 16 provides the following: The method of embodiment 15, wherein the promoter is an EF-1α promoter. Aspect 17 provides the following: The method of embodiment 14, wherein the promoter is a tissue-specific promoter. Aspect 18 provides the following: The method of embodiment 17, wherein the tissue-specific promoter is specific to renal tubular proximal cells. Aspect 19 provides the following: The method of embodiment 18, wherein the tissue specific promoter is selected from the group consisting of Npt2a and Sgt12. Aspect 20 provides the following: The method of embodiment 13, wherein the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1. Aspect 21 provides the following: A pharmaceutical composition comprising a nucleic acid vector encoding a CLCN5 protein and a pharma- ceutically acceptable carrier. Aspect 22 provides the following: 22. The pharmaceutical composition of embodiment 21, wherein the nucleic acid vector is a lentiviral vector. Aspect 23 provides the following: 23. The pharmaceutical composition of embodiment 22, wherein the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:1. Aspect 24 provides the following: A mouse model of type 1 Dent disease, wherein the mouse comprises one or more mutations in the CLCN5 gene of the mouse. Aspect 25 provides the following: 25. The mouse model of embodiment 24, wherein said one or more mutations are a deletion. Aspect 26 provides the following: 26. The mouse model of embodiment 25, wherein said deletion affects exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11 of the CLCN5 gene. Aspect 27 provides the following: The mouse model of embodiment 24, wherein said one or more CLCN5 mutations result in a non-functional CLCN5 protein. Aspect 28 provides the following: 25. The mouse model of embodiment 24, wherein breeding of the experimental animals requires sires and dams of different strains. Aspect 29 provides the following: The mouse model of embodiment 28, wherein the female parent is heterozygous for a CLCN5 mutation and the male parent is wild-type. Aspect 30 provides the following: The mouse model of embodiment 28, wherein the sire is of an FVB background. Aspect 31 provides the following: 29. The mouse model of embodiment 28, wherein the dam is of a C57BL / 6 background.

[0170] Other Aspects The recitation of a list of elements in any definition of a variable herein includes that definition of the variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0171] The disclosures of any and all patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition comprising a nucleic acid vector encoding a CLCN5 protein and a pharma- ceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the nucleic acid vector is a lentiviral vector.

3. The pharmaceutical composition of claim 1, wherein the nucleic acid vector is operably linked to a promoter that drives expression of a CLCN5 protein.

4. The pharmaceutical composition of claim 3, wherein the promoter is a constitutive promoter.

5. 5. The pharmaceutical composition of claim 4, wherein the promoter is an EF-1α promoter.

6. The pharmaceutical composition of claim 3, wherein the promoter is a tissue-specific promoter.

7. 7. The pharmaceutical composition of claim 6, wherein the tissue-specific promoter is specific to renal tubular proximal cells.

8. 8. The pharmaceutical composition of claim 7, wherein the tissue-specific promoter is selected from the group consisting of Npt2a and Sgt12.

9. The pharmaceutical composition of claim 2, wherein the lentiviral vector is encoded by the nucleic acid sequence shown in SEQ ID NO:

1.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, for treating Dent's disease in a subject in need thereof.

11. 11. The pharmaceutical composition of claim 10, administered to the subject, wherein said administration locally delivers said nucleic acid vector to the kidney.

12. The pharmaceutical composition of claim 10, which is administered locally to the kidney by retrograde ureteral injection.

13. A pharmaceutical composition according to any one of claims 1 to 9 for correcting a mutation in a CLCN5 gene in a cell, wherein the CLCN5 protein is a functional CLCN5 protein.

14. A mouse model of type 1 Dent disease, wherein the mouse comprises one or more mutations in the CLCN5 gene of the mouse.

15. The mouse model of claim 14, wherein the one or more mutations are deletions.

16. The mouse model of claim 15, wherein the deletion affects exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11 of the CLCN5 gene.

17. The mouse model of claim 14, wherein the one or more CLCN5 mutations result in a non-functional CLCN5 protein.

18. The mouse model of claim 14, wherein breeding of the experimental animals requires males and females of different strains.

19. The mouse model of claim 18, wherein the female parent is heterozygous for a CLCN5 mutation and the male parent is wild type.

20. The mouse model of claim 18, wherein the sire is of an FVB background.

21. The mouse model of claim 18, wherein the dam is of a C57BL / 6 background.

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