Treatment of renal diseases with a combination of angiopoietin-like 3 (ANGPTL3) inhibitor and solute carrier family 5 member 2 (SLC5A2) inhibitor

Abstract

The present disclosure provides methods for treating a subject with renal disease or preventing a subject from developing renal disease, as well as methods for identifying a subject at increased risk for developing renal disease, by administering an angiopoietin-like 3 (ANGPTL3) inhibitor and a solute carrier family 5 member 2 (SLC5A2) inhibitor.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as an XML file entitled 381203963SEQ, created on November 9, 20223, and is 3,455,297 bytes in size. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure broadly relates to treatment of subjects with renal disease with angiopoietin-like 3 (ANGPTL3) inhibitors and solute carrier family 5 member 2 (SLC5A2) inhibitors, and methods for identifying subjects at increased risk of developing renal disease. [Background technology]

[0003] In the United States, based on data from the 1999–2006 National Health and Nutrition Examination Survey (NHANES), an estimated 11.1% (22.4 million) of adults aged 20 years or older have stages 1–3 chronic kidney disease (CKD). Additionally, 800,000 U.S. adults aged 20 years or older have stage 4 CKD, and over 300,000 have stage 5 CKD and are receiving hemodialysis. Analysis of NHANES data between 1988–1994 and 1999–2004 suggests that the prevalence of CKD has increased for each CKD stage, with a particular increase in the prevalence of individuals classified as stage 3 CKD. The number of patients with stage 5 CKD requiring dialysis is also increasing. It is estimated that by 2015, more than 700,000 people will have end-stage renal disease (ESRD). Although CKD can be caused by primary kidney disease (e.g., glomerular disease, tubulointerstitial disease, obstructive disease, and polycystic kidney disease), in the majority of patients with CKD, kidney damage is associated with other conditions, such as diabetes and hypertension. In 2008, 48% of Medicare patients with CKD, excluding those with ESRD, had diabetes, 91% had hypertension, and 46% had atherosclerotic heart disease. Other risk factors for CKD include age, obesity, family history, and ethnicity. CKD is associated with many adverse health outcomes.

[0004] A glomerular filtration rate (GFR) of 90 mL / min or higher (Stage 1) is normal in most healthy people. This stage of CKD usually presents with few symptoms. A GFR of 60–89 mL / min (Stage 2) may be normal in some patients, such as elderly individuals or infants, in the absence of kidney damage. A GFR between 60–89 mL / min for more than three months, along with concurrent kidney damage, is a sign of early CKD. Symptoms are usually absent at this stage. A patient with a GFR of 30–59 mL / min (Stage 3) indicates moderate CKD and is likely to develop anemia, early bone disease, or hypertension, and should be seen by a nephrology specialist. A GFR between 15–29 mL / min (Stage 4) indicates that the patient has severe CKD and is likely to require dialysis or a kidney transplant in the future. A GFR of 15 mL / min or lower (Stage 5) indicates that the patient has chronic CKD and is at risk of ESRD. The kidneys have lost almost all ability to function effectively at this stage, and they will require dialysis or a kidney transplant to survive.

[0005] The ANGPTL3 gene encodes a member of a family of secreted proteins that function in angiogenesis. The encoded protein, primarily expressed in the liver, is further processed into an N-terminal coiled-coil domain-containing chain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain may be involved in angiogenesis. Mutations in this gene cause familial hypobetalipoproteinemia type 2.

[0006] The SLC5A2 gene encodes a low-affinity, high-capacity Na(+) / glucose cotransporter, which is located in the S1 segment of the early proximal tubule and has a Na(+) to glucose coupling ratio of 1:1. It is the major reabsorption mechanism for D-glucose in the kidney. Summary of the Invention

[0007] The present disclosure provides a method of treating a subject having or at risk of developing renal disease, the method comprising administering to the subject an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

[0008] The present disclosure also provides a method of treating a subject with a renal disease therapeutic agent by administering the renal disease therapeutic agent, wherein the subject has or is at risk of developing renal disease, the method comprising determining whether the subject has an ANGPTL3 variant nucleic acid molecule and whether the subject has an SLC5A2 variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule, and administering or having performed a renal disease therapeutic agent to a subject that is homozygous for both an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule. or administering or continuing to administer a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who is i) heterozygous for both an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule, or ii) heterozygous for one of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule and a reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or iii) an ANGPTL3 reference and an SLC5A2 reference, wherein the presence of a genotype having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule indicates that the subject has a reduced risk of developing kidney disease.

[0009] The present disclosure also provides a method for identifying a subject at increased risk of developing kidney disease, the method comprising determining or having determined the presence or absence of an ANGPTL3 variant nucleic acid molecule and determining or having determined the presence or absence of an SLC5A2 variant nucleic acid molecule in a biological sample obtained from the subject, wherein the subject is at increased risk of developing kidney disease if the subject is the ANGPTL3 reference and the SLC5A2 reference, and the subject is at decreased risk of developing kidney disease if the subject is heterozygous or homozygous for the ANGPTL3 variant nucleic acid molecule and heterozygous or homozygous for the SLC5A2 variant nucleic acid molecule, or if the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and the reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule.

[0010] The present disclosure also provides a renal disease therapeutic agent for use in treating or preventing renal disease in a subject having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule.

[0011] The present disclosure also provides ANGPTL3 inhibitors and SLC5A2 inhibitors for use in treating or preventing kidney disease in a subject: i) heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule; ii) heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule; or iii) heterozygous for one of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule and a reference for the other of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule. DETAILED DESCRIPTION OF THE INVENTION

[0012] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0013] Unless expressly stated otherwise, any method or aspect described herein is in no way intended to be construed as requiring its steps to be performed in a particular order. Thus, unless specifically defined by a method claim in the claims or description, no order is intended to be imposed in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or workflow, general meanings derived from grammatical construction or punctuation, or the number or type of aspects described herein.

[0014] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0015] As used herein, the term "about" means that the recited numerical value is an approximation and that small variations would not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0016] As used herein, the term "comprising" may be replaced in certain embodiments with "consisting of" or "consisting essentially of," as desired.

[0017] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. A reference to one strand of a nucleic acid also refers to its complementary strand.

[0018] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, livestock (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under a physician's care.

[0019] According to the present disclosure, it has been found that rare NGPTL3 variant nucleic acid molecules and rare SLC5A2 variant nucleic acid molecules have independent effects on reducing the risk of developing kidney disease. Both rare NGPTL3 variant nucleic acid molecules and rare SLC5A2 variant nucleic acid molecules result in an increase in estimated glomerular filtration rate (eGFR). It was expected that the two genes, each of which results in an increase in eGFR, may function through the same mechanism / pathway. If this is the case, it is expected that the effect of a variant in one gene will depend on whether a variant in the second gene is also present. If this is the case, it would be expected that the effect of one gene will have a different degree of effect when combined with the effect of the other gene. Surprisingly, the increase in eGFR associated with rare NGPTL3 variant nucleic acid molecules was independent of the increase in eGFR associated with rare SLC5A2 variant nucleic acid molecules. Thus, a subject who is heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule would benefit from being treated with inhibitors of two different biological pathways (e.g., with an ANGPTL3 inhibitor and an SLC5A2 inhibitor), such that renal disease is inhibited or prevented, its symptoms are alleviated or prevented, and / or the onset of symptoms is suppressed or prevented. It is also contemplated that such subjects with renal disease may be further treated with a renal disease therapeutic agent.

[0020] For the purpose of this disclosure, any particular subject (e.g., human) can be classified as having one of three ANGPTL3 genotypes: i) ANGPTL3 reference, ii) heterozygous for ANGPTL3 variant nucleic acid molecule, or iii) homozygous for ANGPTL3 variant nucleic acid molecule.If a subject does not have a copy of ANGPTL3 variant nucleic acid molecule, the subject is ANGPTL3 reference.If a subject has a single copy of ANGPTL3 variant nucleic acid molecule, the subject is heterozygous for ANGPTL3 variant nucleic acid molecule.

[0021] In any of the embodiments described herein, the ANGPTL3 variant nucleic acid molecule can be any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule generated from an mRNA molecule) encoding an ANGPTL3 variant polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. A subject with an ANGPTL3 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for ANGPTL3. In some embodiments, the ANGPTL3 variant nucleic acid molecule results in reduced or abnormal expression or activity of ANGPTL3 mRNA or polypeptide. In some embodiments, the ANGPTL3 variant nucleic acid molecule is associated with a reduced in vitro response to an ANGPTL3 ligand compared to a reference ANGPTL3. In some embodiments, the ANGPTL3 variant nucleic acid molecule is a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide. In some embodiments, the NGPTL3 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the NGPTL3 variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the ANGPTL3 variant nucleic acid molecule has a variation in the coding region. In some embodiments, the ANGPTL3 variant nucleic acid molecule causes or is predicted to cause premature cleavage of the ANGPTL3 polypeptide compared to a reference ANGPTL3. In some embodiments, the ANGPTL3 variant nucleic acid molecule is a variant predicted by an in vitro prediction algorithm, such as Polyphen, SIFT, or a similar algorithm, to impair protein function (and thus be protective in humans in this case).In some embodiments, the ANGPTL3 variant nucleic acid molecule is a variant that causes or is predicted to cause a non-synonymous amino acid substitution in the ANGPTL3 nucleic acid molecule, and whose allele frequency is less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the ANGPTL3 variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%, or 1 in 1,000 alleles), or any splice site, stop gain, start loss, stop loss, frameshift, in-frame indel, or other frameshift ANGPTL3 variant.

[0022] For the purposes of this disclosure, any particular subject, e.g., a human, can be categorized as having one of three SLC5A2 genotypes: i) SLC5A2 reference, ii) heterozygous for an SLC5A2 variant nucleic acid molecule, or iii) homozygous for an SLC5A2 variant nucleic acid molecule. If a subject does not have a copy of an SLC5A2 variant nucleic acid molecule, the subject is SLC5A2 reference. If a subject has a single copy of an SLC5A2 variant nucleic acid molecule, the subject is heterozygous for an SLC5A2 variant nucleic acid molecule.

[0023] In some embodiments, the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, and is a reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule.

[0024] In any of the embodiments described herein, the SLC5A2 variant nucleic acid molecule can be any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule generated from an mRNA molecule) encoding an SLC5A2 variant polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. A subject with an SLC5A2 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for SLC5A2. In some embodiments, the SLC5A2 variant nucleic acid molecule results in reduced or abnormal expression or activity of SLC5A2 mRNA or polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is associated with a reduced in vitro response to an SLC5A2 ligand compared to a reference SLC5A2. In some embodiments, the SLC5A2 variant nucleic acid molecule is a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the SLC5A2 variant nucleic acid molecule comprises a single nucleotide polymorphism (SNP). In some embodiments, the SLC5A2 variant nucleic acid molecule has a variation in the coding region. In some embodiments, the SLC5A2 variant nucleic acid molecule causes or is predicted to cause premature truncation of the SLC5A2 polypeptide compared to a reference SLC5A2. In some embodiments, the SLC5A2 variant nucleic acid molecule is a variant predicted by an in vitro prediction algorithm, such as Polyphen, SIFT, or a similar algorithm, to impair protein function (and thus, in this case, be protective in humans).In some embodiments, the SLC5A2 variant nucleic acid molecule is a variant that causes or is predicted to cause a nonsynonymous amino acid substitution in an SLC5A2 nucleic acid molecule, and whose allele frequency is less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the SLC5A2 variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%, or 1 in 1,000 alleles), or any splice site, stop-gain, start-loss, stop-loss, frameshift, in-frame indel, or other frameshift SLC5A2 variant.

[0025] For subjects who have been genotyped or determined to be ANGPTL3 reference and SLC5A2 reference, such subjects are at increased risk of developing kidney disease. For subjects who have been genotyped or determined to be i) either heterozygous for an ANGPTL3 reference or an ANGPTL3 variant nucleic acid molecule, and ii) heterozygous for an SLC5A2 reference or an SLC5A2 variant nucleic acid molecule, such subjects may be treated with an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

[0026] In any of the embodiments described herein, the subject whose kidney disease is prevented by administering an ANGPTL3 inhibitor and an SLC5A2 inhibitor may be any individual at risk of developing kidney disease, including, but not limited to, a subject with a genetic predisposition to developing kidney disease. Additional risk factors include, but are not limited to, diabetes, hypertension, obesity, excessive salt intake, age, smoking, excessive alcohol consumption, heavy metal exposure, hyperlipidemia, and the presence of autoimmune disease. Furthermore, in some embodiments, any subject may be at risk of developing kidney disease. In some embodiments, administering an ANGPTL3 inhibitor and an SLC5A2 inhibitor may be performed to prevent the development of additional kidney disease in a subject who already has kidney disease.

[0027] In any of the embodiments described herein, the ANGPTL3 polypeptide may be any ANGPTL3 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. In any of the embodiments described herein, the SLC5A2 polypeptide may be any SLC5A2 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function.

[0028] In any of the embodiments described herein, the ANGPTL3 variant nucleic acid molecule (a genomic nucleic acid molecule, an mRNA molecule produced therefrom, or a cDNA molecule produced from the mRNA molecule) can comprise a variation at positions 62,597,520 to 62,606,313 on chromosome 1, using the nucleotide sequence of an ANGPTL3 reference genomic nucleic acid molecule as the reference sequence (see ENST00000371129.4 annotated in the Ensembl database (URL: World Wide Web, "useast.ensembl.org / Homo_sapiens / Transcript / Summary?db=core;g=ENSG00000132855;r=1:62597520-62606313;t=ENST00000371129").

[0029] The sequence provided in ENST00000371129.4 for ANGPTL3 genomic nucleic acid molecule is only exemplary sequence.Other sequences for ANGPTL3 genomic nucleic acid molecule are also possible.Exemplary ANGPTL3 variant nucleic acid molecules include but are not limited to those listed in Table 6.

[0030] In any of the embodiments described herein, the SLC5A2 variant nucleic acid molecule (a genomic nucleic acid molecule, an mRNA molecule produced therefrom, or a cDNA molecule produced from the mRNA molecule) can comprise a variation at positions 31,483,123 to 31,490,769 on chromosome 16, using the nucleotide sequence of an SLC5A2 reference genomic nucleic acid molecule as the reference sequence (see ENST00000330498.4 annotated in the Ensembl database (URL: World Wide Web, "useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000140675;r=16:31483002-31490860;transcript=ENST00000330498.4").

[0031] The sequence provided in ENST00000330498.4 for the SLC5A2 genomic nucleic acid molecule is merely an exemplary sequence. Other sequences for the SLC5A2 genomic nucleic acid molecule are also possible. Exemplary SLC5A2 variant nucleic acid molecules include, but are not limited to, those listed in 6.

[0032] Any one or more (i.e., any combination) of the ANGPTL3 variant nucleic acid molecules and SLC5A2 variant nucleic acid molecules described herein can be used in any of the methods described herein to determine whether a subject has an increased or decreased risk of developing kidney disease. A particular combination of variants can form a mask used in statistical analysis of a particular correlation between ANGPTL3 and SLC5A2 and an increased or decreased risk of developing kidney disease. In some embodiments, the mask used in statistical analysis of a particular correlation between ANGPTL3 and SLC5A2 and an increased or decreased risk of developing kidney disease can exclude one or more of these ANGPTL3 variant nucleic acid molecules and / or SLC5A2 variant nucleic acid molecules described herein.

[0033] In any of the embodiments described herein, the subject may have kidney disease. In any of the embodiments described herein, the subject may be at risk of developing kidney disease. In any of the embodiments described herein, the kidney disease is chronic kidney disease, diabetic kidney disease, kidney stones, chronic glomerulonephritis, nephronophthisis, chronic interstitial nephritis, and / or nephrosclerosis. In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is diabetic kidney disease. In some embodiments, the kidney disease is kidney stones. In some embodiments, the kidney disease is chronic glomerulonephritis. In some embodiments, the kidney disease is nephronophthisis. In some embodiments, the kidney disease is chronic interstitial nephritis. In some embodiments, the kidney disease is nephrosclerosis.

[0034] Other kidney diseases include acquired cystic disease, acute (post-infectious) glomerulonephritis, acute infectious interstitial nephritis, acute interstitial nephritis, acute pyelonephritis, acute renal failure, acute transplant failure, acute tubular necrosis, adult polycystic kidney disease, AL amyloid, analgesic nephropathy, ANCA-associated vasculitis, anti-glomerular basement membrane disease (Goodpasture's syndrome), antibody-mediated renal transplant rejection, asymptomatic hematuria, asymptomatic proteinuria, atypical hemolytic uremic syndrome, autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, BK virus-associated nephropathy, Bence-Jones nephropathy, benign familial nephropathy, hematuria, benign nephrosclerosis, and Atheroembolism, bilateral cortical necrosis, C3 glomerulonephritis, cardiac surgery-related acute kidney injury, chronic allograft nephropathy, chronic glomerulonephritis, chronic interstitial nephritis, chronic pyelonephritis, chronic renal failure, chronic graft failure, circulating immune complex nephritis, contrast nephropathy, crescentic glomerulonephritis, cryoglobulinemia, cystic renal dysplasia, delayed graft function, hyperdensity syndrome, diabetic glomerulosclerosis, diabetic nephropathy, dialysis-related cystic disease, drug-induced (allergic) acute interstitial nephritis, ectopic kidney, eosinophilic granulomatosis with polyangiitis, Fabry disease, familial juvenile nephronophthisis-medullary cystic disease complex, focal segmental glomerulosclerosis (segmental glomerulosclerosis) nodular hyalinosis), glomerular cyst disease, glomerulonephritis, glomerulonephritis associated with bacterial endocarditis, glomerulosclerosis, granulomatosis with polyangiitis, hemolytic uremic syndrome, Henoch-Schönlein purpura, glomerulonephritis associated with hepatitis, hereditary nephritis (Alport syndrome), human immunodeficiency virus-associated nephropathy, horseshoe kidney, hydronephrosis, hyperoxaluria, hypertensive nephropathy, IgA nephropathy, infantile polycystic kidney disease, ischemic acute tubular necrosis, light chain deposition disease, lupus nephritis, malignant nephrosclerosis, medullary cystic disease, membranous proliferative (mesangial capillary) glomerulonephritis, membranous glomerulonephritis, membranous nephropathy, mesangial proliferative glomeruli Nephritis (including Berger's disease), microscopic polyangiitis, minimal change glomerular disease, nephritic syndrome, nephroblastoma (Wilms' tumor), nephronophthisis (medullary cystic disease complex), pigmented nephropathy, plasma cell dyscrasia (monoclonal immunoglobulin-induced nephropathy), polyarteritis nodosa, polycystic kidney disease, proteinuria, pyelonephritis, rapidly progressive (crescentic) glomerulonephritis, renal agenesis, renal amyloidosis, renal cell carcinoma, renal hypoplasia, renal dysplasia, renal hypoplasia, renal infection, renal osteodystrophy, kidney stones (urolithiasis), renal tubular acidosis, renal vasculitis, renovascular hypertension, scleroderma (progressive systemic sclerosis),These conditions include, but are not limited to, secondary acquired glomerulonephritis, sepsis-associated acute kidney injury, simple renal cysts, systemic lupus erythematosus, T-cell-mediated renal transplant rejection, thin basement membrane nephropathy, thrombotic microangiopathy, thrombotic thrombocytopenic purpura, toxic acute tubular necrosis, tubular defects, tubulointerstitial disease in multiple myeloma, uric acid nephropathy, urinary tract obstruction, and vasculitis.

[0035] Symptoms of chronic kidney disease include, but are not limited to, nausea, vomiting, loss of appetite, fatigue and weakness, sleep disturbances, changes in urination, decreased mental acuity, muscle cramps and spasms, swelling of the feet and ankles, persistent itching, chest pain, fluid buildup around the endocardium of the heart, shortness of breath, fluid buildup in the lungs, and high blood pressure (hypertension) that is difficult to control.

[0036] Symptoms of kidney stones include, but are not limited to, severe, sharp pain in the side and back below the ribs, pain radiating to the lower abdomen and groin, pain that comes in waves and fluctuates in intensity, pain or burning during urination, pink, red or brown urine, cloudy or foul-smelling urine, a persistent need to urinate, urinating more frequently or in smaller amounts than usual, nausea and vomiting, and fever and chills if an infection is present.

[0037] Symptoms of chronic glomerulonephritis include, but are not limited to, pink or cola-colored urine (hematuria) from red blood cells in the urine, foamy urine due to excess protein (proteinuria), high blood pressure (hypertension), and fluid retention (edema), evident as swelling in the face, hands, feet, and abdomen.

[0038] Symptoms of nephronophthisis include, but are not limited to, increased urine production (polyuria), excessive thirst (polydipsia), general weakness, and extreme tiredness (fatigue).

[0039] Symptoms of chronic interstitial nephritis include, but are not limited to, blood in the urine, fever, increased or decreased urine output, mental status changes (drowsiness, confusion, coma), nausea, vomiting, rash, swelling in any area of ​​the body, and weight gain (from retained fluid).

[0040] Symptoms of nephrosclerosis include, but are not limited to, blurred vision, blood in the urine, weight loss, and accumulation of urea and other nitrogenous waste products in the blood (a condition known as uremia).

[0041] The present disclosure provides methods of treating a subject having or at risk of developing kidney disease, the method comprising administering to the subject an ANGPTL3 inhibitor and an SLC5A2 inhibitor. In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is diabetic kidney disease. In some embodiments, the kidney disease is kidney stones. In some embodiments, the kidney disease is chronic glomerulonephritis. In some embodiments, the kidney disease is nephronophthisis. In some embodiments, the kidney disease is chronic interstitial nephritis. In some embodiments, the kidney disease is nephrosclerosis.

[0042] In some embodiments, the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the ANGPTL3 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within an ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of ANGPTL3 polypeptide in cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an antisense molecule that hybridizes to an ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of ANGPTL3 polypeptide in cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an siRNA that hybridizes to an ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of ANGPTL3 polypeptide in cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an shRNA that hybridizes to an ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces expression of an ANGPTL3 polypeptide in cells in a subject.

[0043] In some embodiments, an ANGPTL3 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 1 to 325. In some embodiments, an ANGPTL3 siRNA molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 326 to 1189 (sense and antisense strands are presented sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 326 and the corresponding antisense strand is SEQ ID NO: 327; the sense strand is, e.g., SEQ ID NO: 1188 and the corresponding antisense strand is SEQ ID NO: 1189, etc.). In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences (sense and antisense strands) set forth in U.S. Patent No. 10,995,335 and PCT Publication No. WO2019 / 055633, which are incorporated herein by reference in their entireties.

[0044] In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences (sense and antisense strands) set forth in U.S. Patent No. 10,875,884 and PCT Publication Nos. WO2015 / 168589, WO2015 / 100394, and WO2011 / 085271, which are incorporated herein by reference in their entireties.

[0045] In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences (sense and antisense strands) set forth in U.S. Patent Nos. 10,570,393 and 10,337,010, and PCT Publication Nos. WO2016 / 168286 and WO2012 / 177784, which are incorporated herein by reference in their entireties.

[0046] In some embodiments, the ANGPTL3 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s), or a DNA-binding protein that binds to the recognition sequence within the ANGPTL3 genomic nucleic acid molecule. The recognition sequence can be located within the coding region of the ANGPTL3 gene or within a regulatory region that affects gene expression. The recognition sequence of the DNA-binding protein or nuclease agent can be located within an intron, exon, promoter, enhancer, regulatory region, or any non-protein-coding region. The recognition sequence can encompass or be adjacent to the start codon of the ANGPTL3 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can delete the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0047] Nuclease agents and DNA-binding proteins suitable for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but examples include recognition sequences that are about 30 to about 36 bp for zinc finger proteins or ZFN pairs, about 15 to about 18 bp for each ZFN, about 36 bp for TALE proteins or TALENs, and about 20 bp for CRISPR / Cas guide RNAs.

[0048] In some embodiments, a CRISPR / Cas system can be used to modify an ANGPTL3 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of an ANGPTL3 nucleic acid molecule.

[0049] Cas proteins generally contain at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins may also contain a nuclease domain (e.g., a DNase or RNase domain), a DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type Cpf1 protein (e.g., FnCpf1). Cas proteins may have full cleavage activity to generate double-strand breaks in ANGPTL3 genomic nucleic acid molecules, or may be nickases that generate single-strand breaks in ANGPTL3 genomic nucleic acid molecules. Further examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (Cas B), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified forms thereof. In some embodiments, a Cas system, such as Cas12a, can have multiple gRNAs encoded by a single crRNA. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas protein can be combined or fused with cleavage domain, epigenetic modification domain, transcription activation domain or transcription repressor domain.Cas protein can be provided in any form.For example, Cas protein can be provided in the form of protein, for example, the Cas protein complexed with gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, that encodes the Cas protein.

[0050] In some embodiments, targeted genetic modification of an ANGPTL3 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the ANGPTL3 genomic nucleic acid molecule. The gRNA recognition sequence can include or be adjacent to the start codon of the ANGPTL3 genomic nucleic acid molecule or the stop codon of the ANGPTL3 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or stop codon.

[0051] The gRNA recognition sequence within the target genomic locus in the ANGPTL3 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The canonical PAM sequence is 5'-NGG-3' (where "N" is any nucleobase and is followed by two guanine ("G") nucleobases). The gRNA can deliver Cas9 to any location in the genome for gene editing, but editing cannot occur at any site other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is approximately 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be flanked by a PAM on the 3' end. In some embodiments, the gRNA recognition sequence can be flanked on the 5' end by a PAM. For example, the cleavage site for the Cas protein can be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence on the non-complementary strand can be 5'-NGG-3' (where N is any DNA nucleotide and is immediately 3' of the gRNA recognition sequence on the non-complementary strand of the target DNA). Thus, the PAM sequence on the complementary strand will be 5'-CCN-3' (where N is any DNA nucleotide and is immediately 5' of the gRNA recognition sequence on the complementary strand of the target DNA).

[0052] gRNA is the RNA molecule that binds to Cas protein and targets Cas protein to specific position in ANGPTL3 genomic nucleic acid molecule.Exemplary gRNA is the gRNA that is effective for inducing Cas enzyme to bind to or cut ANGPTL3 genomic nucleic acid molecule, and gRNA comprises the DNA targeting segment that hybridizes with the gRNA recognition sequence in ANGPTL3 genomic nucleic acid molecule.Exemplary gRNA comprises the DNA targeting segment that hybridizes with the gRNA recognition sequence that exists in ANGPTL3 genomic nucleic acid molecule that includes or is close to start codon or stop codon. For example, a gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides away from the start codon, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides away from the stop codon. Suitable gRNAs can comprise about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0053] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target ANGPTL3 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target ANGPTL3 genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can cleave one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the ANGPTL3 genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA will bind.

[0054] This method can result in, for example, an ANGPTL3 genomic nucleic acid molecule in which a region of the ANGPTL3 genomic nucleic acid molecule is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted.Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus in the ANGPTL3 genomic nucleic acid molecule.By contacting the cell with one or more additional gRNAs (for example, a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0055] In some embodiments, the ANGPTL3 inhibitor is a small molecule. In some embodiments, the ANGPTL3 inhibitor is (12mer-)heparin (Gunn et al., J. Biol. Chem., 2021, 296, 1-12) or CAT-2003 (Liu et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 2014, 34, A237). In some embodiments, the ANGPTL3 inhibitor is a vaccine. In some embodiments, the vaccine comprises a peptide corresponding to the LPL inhibitory domain of ANGPTL3. In some embodiments, the vaccine comprises a peptide having an amino acid sequence including amino acids 32-41 of ANGPTL3 (i.e., EPKSRFAMLD, SEQ ID NO: 3738) (Fukami et al., Cell Reports Med., 2021, 100446).

[0056] In some embodiments, the ANGPTL3 inhibitor is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human ANGPTL3. Exemplary antibodies and fragments thereof are disclosed in PCT Publication WO2020 / 243031, which is incorporated herein by reference in its entirety.

[0057] In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to, neutralizes, inhibits, blocks, blocks, abrogates, reduces, or interferes with at least one activity of ANGTPL3, particularly human ANGPTL3 (SEQ ID NO: 3739). Activities of ANGPTL3 that can be neutralized, inhibited, blocked, abrogated, reduced, or interfered with by an antibody or fragment thereof of the present disclosure include, but are not limited to, inhibition of LPL activity, induction of angiogenesis, etc. In some embodiments, the antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with the activity of ANGPTL3 by binding to an epitope of ANGPTL3 that is directly involved in the target activity of ANGPTL3. In some embodiments, an antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with the activity of ANGPTL3 by binding to an epitope of ANGPTL3 that is not directly involved in the target activity of ANGPTL3, but the antibody or fragment that binds thereto sterically or conformationally inhibits, blocks, abrogates, reduces, or interferes with the target activity of ANGPTL3. In some embodiments, an antibody or fragment thereof binds to an epitope of ANGPTL3 that is not directly involved in the target activity of ANGPTL3 (i.e., a non-blocking antibody), but the antibody or fragment that binds thereto results in enhanced clearance of ANGPTL3 from the circulation compared to clearance of ANGPTL3 in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, abrogating, reducing, or interfering with the activity of ANGPTL3. Clearance of ANGPTL3 from the circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with each other for specific binding to ANGPTL3.

[0058] Antibodies (Abs) may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

[0059] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 3740, 3741, 3742, 3743, 3744, 3745, 3746, 3747, 3748, 3749, and 3750, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3740, 3741, 3742, 3744, 3745, 3747, and 3750. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 3744.

[0060] In some embodiments, the antibody or antigen-binding fragment of the antibody comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 3751, 3752, 3753, 3754, 3755, 3756, 3757, 3758, 3759, 3760, and 3761, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In another embodiment, the antibody or antigen-binding portion of the antibody comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3751, 3752, 3753, 3755, 3756, 3758, and 3761. In some embodiments, the antibody or antigen-binding portion comprises a LCVR having the amino acid sequence of SEQ ID NO: 3755.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR and LCVR sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3743 / 3754, 3744 / 3755, 3745 / 3756, 3746 / 3757, 3747 / 3758, 3748 / 3759, 3749 / 3760, and 3750 / 3761. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR and LCVR sequence pair selected from the group consisting of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3744 / 3755, 3745 / 3756, 3747 / 3758, and 3750 / 3761. In some embodiments, the antibody or fragment thereof comprises the HCVR and LCVR sequence pair of SEQ ID NOs: 3744 / 3755.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence selected from the group consisting of SEQ ID NOs: 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, and 3772, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain CDR3 (LCDR3) amino acid sequence selected from the group consisting of SEQ ID NOs: 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, and 3783, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the antibody or fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair comprising SEQ ID NOs: 3762 / 3773, 3763 / 3774, 3764 / 3775, 3765 / 3776, 3766 / 3777, 3767 / 3778, 3768 / 3779, 3769 / 3780, 3770 / 3781, 3771 / 3782, or 3772 / 3783. In some embodiments, the antibody or fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair comprising SEQ ID NOs: 3762 / 3773, 3763 / 3774, 3764 / 3775, 3766 / 3777, 3767 / 3778, 3769 / 3780, or 3772 / 3783. In some embodiments, the antibody or fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair comprising SEQ ID NOs: 3766 / 3777.

[0063] In some embodiments, the antibody or fragment thereof further comprises a heavy chain CDR1 (HCDR1) amino acid sequence selected from the group consisting of SEQ ID NOs: 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, and 3794, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CDR2 (HCDR2) amino acid sequence selected from the group consisting of SEQ ID NOs: 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, and 3805, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and optionally, a heavy chain CDR2 (HCDR2) amino acid sequence selected from the group consisting of SEQ ID NOs: 3806, a light chain CDR1 (LCDR1) amino acid sequence selected from the group consisting of 3807, 3808, 3809, 3810, 3811, 3812, 3813, 3814, 3815, and 3816, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and / or SEQ ID NOs: 3817 (AAS), 3818 (KAS), 3819 (AAS), 3820(KAS), 3821(KAS), 3822(TTS), 3823(PAS), 3824(TAS), 3825(KVS), 3826(VAA), and 3827, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof has an HCDR1 / HCDR2 / HCDR sequence selected from the group consisting of SEQ ID NOs: 3784 / 3795 / 3762, 3785 / 3796 / 3763, 3786 / 3797 / 3764, 3787 / 3798 / 3765, 3788 / 3799 / 3766, 3789 / 3800 / 3767, 3790 / 3801 / 3768, 3791 / 3802 / 3769, 3792 / 3803 / 3770, 3793 / 3804 / 3771, and 3794 / 3805 / 3772. 3806 / 3817 / 3773, 3807 / 3818 / 3774, 3808 / 3819 / 3775, 3809 / 3820 / 3776, 3810 / 3821 / 3777, 3811 / 3822 / 3778, 3812 / 3823 / 3779, 3813 / 3824 / 3780, 3814 / 3825 / 3781, 3815 / 3826 / 3782, and 3816 / 3827 / 3783. In some embodiments, the heavy and light chain CDR amino acid sequences are selected from the group consisting of SEQ ID NOs: 3784 / 3795 / 3762 / 3806 / 3817 / 3773, 3785 / 3796 / 3763 / 3807 / 3818 / 3774, 3786 / 3797 / 3764 / 3808 / 3819 / 3775, 3787 / 3798 / 3765 / 3809 / 3820 / 3776, 3788 / 3799 / 3766 / 3810 / 3821 / 3777, 3789 / 3800 / 3 and 3794 / 3805 / 3772 / 3816 / 3827 / 3783.In some embodiments, the heavy and light chain CDR amino acid sequences comprise a combination of CDR sequences selected from the group consisting of SEQ ID NOs: 3784 / 3795 / 3762 / 3806 / 3817 / 3773, 3785 / 3796 / 3763 / 3807 / 3818 / 3774, 3786 / 3797 / 3764 / 3808 / 3819 / 3775, 3788 / 3799 / 3766 / 3810 / 3821 / 3777, 3789 / 3800 / 3767 / 3811 / 3822 / 3778, 3791 / 3802 / 3769 / 3813 / 3824 / 3780, or 3794 / 3805 / 3772 / 3816 / 3827 / 3783. In some embodiments, the heavy and light chain CDR amino acid sequences comprise a combination of the CDR sequences of SEQ ID NOs: 3788 / 3799 / 3766 / 3810 / 3821 / 3777.

[0065] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3 comprises heavy chain and light chain CDR domains contained within an HCVR / LCVR pair selected from the group consisting of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3743 / 3754, 3744 / 3755, 3745 / 3756, 3746 / 3757, 3747 / 3758, 3748 / 3759, 3749 / 3760, and 3750 / 3761. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be applied to identify CDRs within specific HCVR and / or LCVR amino acid sequences disclosed herein. Conventional definitions that can be applied to identify CDR boundaries include the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol., 1997, 273, 927-948; and Martin et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 9268-9272. Public databases are also available for identifying CDR sequences within antibodies. In some embodiments, the antibody or fragment thereof comprises the CDR sequences contained within the HCVR and LCVR pair of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3744 / 3755, 3745 / 3756, 3747 / 3758, or 3750 / 3761. In some embodiments, the antibody or fragment thereof comprises the CDR sequences contained within the HCVR and LCVR pair of SEQ ID NOs: 3744 / 3755.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof competes for specific binding to ANGPTL3 with an antibody or antigen-binding fragment comprising heavy and light chain CDR sequences contained in the HCVR / LCVR sequence pair of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3743 / 3754, 3744 / 3755, 3745 / 3756, 3746 / 3757, 3747 / 3758, 3748 / 3759, 3749 / 3760, or 3750 / 3761. In some embodiments, the antibody or antigen-binding fragment thereof competes for specific binding to ANGPTL3 with an antibody or fragment thereof comprising the HCVR / LCVR sequence pair of SEQ ID NOs: 3744 / 3755. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: 3784 / 3795 / 3762 / 3806 / 3817 / 3773, 3785 / 3796 / 3763 / 3807 / 3818 / 3774, 3786 / 3797 / 3764 / 3808 / 3819 / 3775, 3787 / 3798 / 3765 / 3809 / 3820 / 3776, 3788 / 3799 / 3766 / 3810 / 3821 / 3777, 3789 / 3800 / 3767 / 3811 / 3822 / 3778, 379 It competes for specific binding to ANGPTL3 with an antibody or antigen-binding fragment thereof comprising a combination of heavy chain and light chain CDR sequences selected from the group consisting of: 0 / 3801 / 3768 / 3812 / 3823 / 3779, 3791 / 3802 / 3769 / 3813 / 3824 / 3780, 3792 / 3803 / 3770 / 3814 / 3825 / 3781, 3793 / 3804 / 3771 / 3815 / 3826 / 3782 and 3794 / 3805 / 3772 / 3816 / 3827 / 3783. In some embodiments, the antibody or antigen-binding fragment thereof competes for specific binding to ANGPTL3 with an antibody or fragment thereof comprising a combination of heavy and light chain CDR sequences of SEQ ID NOs: 3788 / 3799 / 3766 / 3810 / 3821 / 3777.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on ANGPTL3 recognized by an antibody or fragment thereof comprising heavy and light chain CDR sequences from the HCVR / LCVR sequence pair of SEQ ID NOs: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3743 / 3754, 3744 / 3755, 3745 / 3756, 3746 / 3757, 3747 / 3758, 3748 / 3759, 3749 / 3760, or 3750 / 3761. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on ANGPTL3 recognized by an antibody or fragment thereof comprising the HCVR / LCVR sequence pair of SEQ ID NOs: 3744 / 3755. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of 3784 / 3795 / 3762 / 3806 / 3817 / 3773, 3785 / 3796 / 3763 / 3807 / 3818 / 3774, 3786 / 3797 / 3764 / 3808 / 3819 / 3775, 3787 / 3798 / 3765 / 3809 / 3820 / 3776, 3788 / 3799 / 3766 / 3810 / 3821 / 3777, 3789 / 3800 / 3767 / 3811 / 3822 / 3778, 3790 / 380 1 / 3768 / 3812 / 3823 / 3779, 3791 / 3802 / 3769 / 3813 / 3824 / 3780, 3792 / 3803 / 3770 / 3814 / 3825 / 3781, 3793 / 3804 / 3771 / 3815 / 3826 / 3782, and 3794 / 3805 / 3772 / 3816 / 3827 / 3783. In some embodiments, such an epitope is recognized by an antibody or antigen-binding fragment thereof comprising the combination of heavy and light chain CDR sequences of SEQ ID NOs: 3788 / 3799 / 3766 / 3810 / 3821 / 3777.

[0068] In some embodiments, the isolated anti-ANGPTL3 antibody or antigen-binding fragment thereof binds to an epitope located within the N-terminal coiled-coil region at residues 17-209 of SEQ ID NO: 3739 and neutralizes, inhibits, abrogates, reduces, or interferes with at least one activity of ANGPTL3. In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to an epitope located within the N-terminal coiled-coil region of ANGPTL3 (SEQ ID NO: 3739) and neutralizes, inhibits, abrogates, reduces, or interferes with at least one activity of ANGPTL3, provided that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 3828 (corresponding to residues Glu32 to Leu57 of ANGPTL3 of SEQ ID NO: 3739). In some embodiments, the antibody or fragment thereof specifically binds to an epitope within residues 17-200, 17-100, 17-70, 17-65, 17-60, 17-57, or 17-50 of ANGPTL3 (SEQ ID NO: 3739), provided that, optionally, the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 3828. In some embodiments, the antibody or fragment thereof specifically binds to an epitope within residues 40-200, 40-100, 40-70, 50-200, 50-100, 50-70, 58-200, 58-100, 58-70, 58-68, or 61-66 of ANGPTL3 (SEQ ID NO: 3739), provided that, optionally, the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 3828. In some embodiments, the antibody or antibody fragment binds to an epitope that may include one or more of the listed epitopes or residues within the N-terminal coil region of ANGPTL3, provided that, optionally, the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 3828.

[0069] In some embodiments, the antibody or fragment thereof comprises an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3829, 3830, 3831, 3832, 3833, 3834, 3835, 3836, 3837, 3838, and 3839, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto. In some embodiments, the antibody or fragment thereof comprises an HCVR encoded by the nucleic acid sequence of SEQ ID NO: 3829, 3830, 3831, 3833, 3834, 3836, or 3839. In some embodiments, the antibody or fragment thereof comprises an HCVR encoded by the nucleic acid sequence of SEQ ID NO: 3833.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3840, 3841, 3842, 3843, 3844, 3845, 3846, 3847, 3848, 3849, and 3850, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto. In some embodiments, the antibody or fragment thereof comprises a LCVR encoded by the nucleic acid sequence of SEQ ID NO: 3840, 3841, 3842, 3844, 3845, 3847, or 3850. In some embodiments, the antibody or fragment thereof comprises a LCVR encoded by the nucleic acid sequence of SEQ ID NO: 3844.

[0071] In some embodiments, the antibody or fragment thereof comprises an HCVR and LCVR (HCVR / LCVR) sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 3829 / 3840, 3830 / 3841, 3831 / 3842, 3832 / 3843, 3833 / 3844, 3834 / 3845, 3835 / 3846, 3836 / 3847, 3837 / 3848, 3838 / 3849, and 3839 / 3850. In some embodiments, the antibody or fragment thereof comprises an HCVR / LCVR sequence pair encoded by the nucleic acid sequence pair of SEQ ID NOs: 3829 / 3840, 3830 / 3841, 3831 / 3842, 3833 / 3844, 3834 / 3845, 3836 / 3847, or 3839 / 3850. In some embodiments, the antibody or fragment thereof comprises the HCVR / LCVR sequence pair encoded by the nucleic acid sequence pair of SEQ ID NOs: 3833 / 3844.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858, 3859, 3860, and 3861, or a substantially identical sequence with at least 90%, at least 95%, at least 98%, or at least 99% homology thereto, and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3862, 3863, 3864, 3865, 3866, 3867, 3868, 3869, 3870, 3871, and 3872, or a substantially identical sequence with at least 90%, at least 95%, at least 98%, or at least 99% homology thereto. In some embodiments, the antibody or fragment thereof comprises an HCDR3 and LCDR3 sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NOs: 3851 / 3862, 3852 / 3863, 3853 / 3864, 3854 / 3865, 3855 / 3866, 3856 / 3867, 3857 / 3868, 3858 / 3869, 3859 / 3870, 3860 / 3871 and 3861 / 3872. In some embodiments, the antibody or fragment thereof comprises an HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NOs: 3851 / 3862, 3852 / 3863, 3853 / 3864, 3855 / 3866, 3856 / 3867, 3858 / 3869 or 3861 / 3872. In some embodiments, the antibody or fragment thereof comprises the HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NOs: 3855 / 3866.

[0073] In some embodiments, the antibody or fragment thereof further comprises an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3873, 3874, 3875, 3876, 3877, 3878, 3879, 3880, 3881, 3882, and 3883, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto; and an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 388 3894, 3895, 3896, 3897, 3898, 3899, 3900, 3901, 3902, 3903, 3904, 3905, 3906, 3907, 3908, 3909, 3910, 3911, 3912, 3913, 3914, 3915, 3916, 3917, 3918, 3919, 3920, 3921, 3922, 3923, 3924, 3925, 3926, 3927, 3928, 3929, 3930, 3931, 3932, 3933, 3934, 3935, 3936, 3937, 3938, 3939, 3940, 3941, 3942, 3943, 3944, 3945, 3946, 3947, 3948, 3949, 3950, 3951, 3952, 3953, 3954, 3955, 3956, 3957, 3958, 3959, 3960, 3961, 3962, 3963, 3964, 3965, 3966, 3967, 3968, 3969, 3970, 3971, 3972, 3973, 3974, 3975, 3976, 3977, 3978, and / or an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3906 (gctgcatcc), 3907 (aaggcgtct), 3908 (gctgcatcc), 3909 (aaggcgtct), 3910 (aaggcgtct), 3911 (actacttcc), 3912 (cctgcatcc), 3913 (actgcatcc), 3914 (aaggtttct), 3915 (gttgcagcc), and 3916, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof has an HCDR1 / HCDR2 / HCDR3 sequence encoded by a combination of nucleotide sequences selected from the group consisting of SEQ ID NOs: 3873 / 3884 / 3851, 3874 / 3885 / 3852, 3875 / 3886 / 3853, 3876 / 3887 / 3854, 3877 / 3888 / 3855, 3878 / 3889 / 3856, 3879 / 3890 / 3857, 3880 / 3891 / 3858, 3881 / 3892 / 3859, 3882 / 3893 / 3860, and 3883 / 3894 / 3861. and / or LCDR1 / LCDR2 / LCDR3 combinations encoded by a combination of nucleotide sequences selected from the group consisting of SEQ ID NOs: 3895 / 3906 / 3862, 3896 / 3907 / 3863, 3897 / 3908 / 3864, 3898 / 3909 / 3865, 3899 / 3910 / 3866, 3900 / 3911 / 3867, 3901 / 3912 / 3868, 3902 / 3913 / 3869, 3903 / 3914 / 3870, 3904 / 3915 / 3871, and 3905 / 3916 / 3872. In some embodiments, the antibody or fragment thereof comprises heavy and light chain CDR sequences encoded by the combination of nucleotide sequences of SEQ ID NOs: 3877 / 3888 / 3855 / 3899 / 3910 / 3866.

[0075] In some embodiments, the anti-ANGPTL3 antibody or antigen-binding fragment thereof is H , D H and J H a heavy chain variable region (HCVR) encoded by a nucleotide sequence segment derived from a germline sequence; and K and J K and a light chain variable region (LCVR) encoded by a nucleotide sequence segment derived from a germline sequence, wherein the HCVR and LCVR are encoded by a nucleotide sequence segment derived from a combination of germline genes selected from the group consisting of: (i) V H 3-43, D H 3-3, 43, V K 1-5 and J K 2;(ii)VH 3-11, D H 1-1, J H 4. V K 1-39 and J K 4;(iii)V H 3-30, D H 1-7, J H 6. V K 1-5 and J K 1;(iv)V H 3-30, D H 1-26, J H 6. V K 1-12 and J K 3;(v)V H 3-30, D H 3-10, J H 6. V K 1-12 and J K 3; and (vi) V H 3-23, D H 3-10, J H 4. V K 1-5 and J K 1.

[0076] In some embodiments, the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (K) of about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less, as measured by a surface plasmon resonance assay (e.g., BIACORE™). D In some embodiments, the antibody specifically binds to ANGPTL3 with a K of about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, or about 50 pM or less. D Shows.

[0077] In some embodiments, the anti-ANGPTL3 antibody has a modified glycosylation pattern. In some applications, modification to remove undesired glycosylation sites, or, for example, removal of fucose moieties to increase antibody-dependent cellular cytotoxicity (ADCC) function, may be useful (see Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation sites can reduce undesired immune responses to therapeutic antibodies or increase antibody affinity. In still other applications, modification of galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

[0078] In some embodiments, the ANGPTL3 antibody is evinacumab.

[0079] In some embodiments, the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, siRNA, and shRNA. Such inhibitory nucleic acid molecules can be designed to target any region of the SLC5A2 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within an SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an SLC5A2 polypeptide in cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an antisense molecule that hybridizes to an SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an SLC5A2 polypeptide in cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an siRNA that hybridizes to an SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of an SLC5A2 polypeptide in cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an shRNA that hybridizes to an SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces expression of an SLC5A2 polypeptide in cells in the subject.

[0080] In some embodiments, the SLC5A2 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 1190 to 1569. In some embodiments, the SLC5A2 siRNA molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 1570 to 3737 (sense and antisense strands are presented sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 1570 and the corresponding antisense strand is SEQ ID NO: 1571; the sense strand is, e.g., SEQ ID NO: 3736 and the corresponding antisense strand is SEQ ID NO: 3737, etc.).

[0081] In some embodiments, the SLC5A2 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at a recognition sequence(s) within the SLC5A2 genomic nucleic acid molecule or a DNA-binding protein that binds to the recognition sequence. The recognition sequence may be located within the coding region of the SLC5A2 gene or within a regulatory region that affects gene expression. The recognition sequence for the DNA-binding protein or nuclease agent may be located within an intron, exon, promoter, enhancer, regulatory region, or any non-protein-coding region. The recognition sequence may include or be adjacent to the start codon of the SLC5A2 gene. For example, the recognition sequence may be located about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon. As another example, two or more nuclease agents may be used, each targeting a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can delete the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0082] Nuclease agents and DNA-binding proteins suitable for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but examples include recognition sequences that are about 30 to about 36 bp for zinc finger proteins or ZFN pairs, about 15 to about 18 bp for each ZFN, about 36 bp for TALE proteins or TALENs, and about 20 bp for CRISPR / Cas guide RNAs.

[0083] In some embodiments, the CRISPR / Cas system can be used to modify the SLC5A2 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the SLC5A2 nucleic acid molecule.

[0084] Cas proteins generally contain at least one RNA recognition or binding domain capable of interacting with a gRNA. Cas proteins may also contain a nuclease domain (e.g., a DNase or RNase domain), a DNA-binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type Cpf1 protein (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-strand breaks in SLC5A2 genomic nucleic acid molecules, or can be nickases that create single-strand breaks in SLC5A2 genomic nucleic acid molecules. Further examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (Cas B), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified forms thereof. In some embodiments, a Cas system, such as Cas12a, can have multiple gRNAs encoded by a single crRNA. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, the Cas protein can be linked to or fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. The Cas protein can be provided in any form.For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA, or in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0085] In some embodiments, targeted genetic modification of an SLC5A2 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus of the SLC5A2 genomic nucleic acid molecule. The gRNA recognition sequence can include or be adjacent to the start codon of the SLC5A2 genomic nucleic acid molecule or the stop codon of the SLC5A2 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start or stop codon.

[0086] The gRNA recognition sequence within the target genomic locus in the SLC5A2 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The canonical PAM is the sequence 5'-NGG-3' (where "N" is any nucleobase and is followed by two guanine ("G") nucleobases). The gRNA can deliver Cas9 to any location in the genome for gene editing, but editing cannot occur at any site other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is approximately 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be flanked on the 3' end by a PAM. In some embodiments, the gRNA recognition sequence can be flanked on the 5' end by a PAM. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence on the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence on the complementary strand of the target DNA.

[0087] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific location in SLC5A2 genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cut SLC5A2 genomic nucleic acid molecule, and this gRNA comprises a DNA targeting segment that hybridizes with the gRNA recognition sequence in SLC5A2 genomic nucleic acid molecule.Exemplary gRNA comprises a DNA targeting segment that hybridizes with the gRNA recognition sequence that exists in SLC5A2 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon. For example, a gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides away from the start codon, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides away from the stop codon. Suitable gRNAs can comprise about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0088] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target SLC5A2 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target SLC5A2 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including a gRNA hybridized with a gRNA recognition sequence and complexed with a Cas protein) can result in one or both strand cleavage within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the SLC5A2 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0089] Such a method can, for example, produce an SLC5A2 genomic nucleic acid molecule in which a region of the SLC5A2 genomic nucleic acid molecule is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted.Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the SLC5A2 genomic nucleic acid molecule.By contacting the cell with one or more additional gRNAs (for example, a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0090] In some embodiments, the SLC5A2 inhibitor targets the SGLT2 polypeptide (a polypeptide encoded by the SLC5A2 gene). In some embodiments, the SLC5A2 inhibitor comprises INVOKANA® (canagliflozin), FORXIGA® (dapagliflozin), JARDIANCE® (empagliflozin), SUGLAT® (ipragliflozin), LUSEFI® (luceogliflozin), or APLEWAY® (tofogliflozin), or any combination thereof. In some embodiments, the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof. In some embodiments, the SLC5A2 inhibitor comprises canagliflozin. In some embodiments, the SLC5A2 inhibitor comprises dapagliflozin. In some embodiments, the SLC5A2 inhibitor comprises empagliflozin. In some embodiments, the SLC5A2 inhibitor comprises ipragliflozin. In some embodiments, the SLC5A2 inhibitor comprises luseogliflozin. In some embodiments, the SLC5A2 inhibitor comprises tofogliflozin.

[0091] Inhibitory nucleic acid molecules can comprise RNA, DNA, or both RNA and DNA. Inhibitory nucleic acid molecules can also be conjugated or fused to heterologous nucleic acid sequences or heterologous labels, for example, in a vector. For example, inhibitory nucleic acid molecules can be present in a vector containing the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence, or as an exogenous donor sequence containing them. Inhibitory nucleic acid molecules can also be conjugated or fused to heterologous labels. Labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. Labels can also be, for example, chemiluminescent substances, metal-containing substances, or enzymes, in which case enzyme-dependent secondary signal generation occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a conjugated molecule so that when the conjugated molecule is subsequently added with a substrate, it can be used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and tested with a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose-binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent, and chemiluminescent substrates, as well as other labels.

[0092] Inhibitory nucleic acid molecules can contain, for example, nucleotides, or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0093] The inhibitory nucleic acid molecule may contain one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide containing modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0094] Nucleotide analogs can also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of the ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications also include -O[(CH) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON[(CH2) n CH3)]2 (where n and m are independently 1 to about 10). Other modifications at the 2' position include, but are not limited to, C 1-10Modifications include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides, and the 5' position of 5'-terminal nucleotides. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0095] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate sites include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarities, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0096] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1 to 7 nucleotides at the 5' and 3' ends each have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides at the 5' and 3' ends each have a 2'-MOE modification. In some embodiments, the first 1 to 7 nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first 5 nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0097] In some embodiments, the siRNA molecule has terminal modification. In some embodiments, the 5'-end of the antisense strand is phosphorylated. In some embodiments, a non-hydrolyzable 5'-phosphate analog, such as 5'-(E)-vinyl-phosphonate, is used.

[0098] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to improve the stability and in vivo bioavailability of siRNA. Non-ester groups (-OH, ═O) in the phosphodiester linkage can be replaced with sulfur, boron, or acetate to yield phosphorothioate, boranophosphate, and phosphonoacetate linkages. Additionally, replacing the phosphodiester group with a phosphotriester can promote cellular uptake of siRNA and retention in serum components by eliminating the negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such modifications include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0099] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases can be substituted with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0100] In some embodiments, siRNA molecules are conjugated to lipids.Lipids can be conjugated to the 5' or 3' end of siRNA to allow them to associate with serum lipoproteins, thereby improving their in vivo bioavailability.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0101] In some embodiments, a representative siRNA has the following formula: Sense:mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N where "N" is the base, "2F" is the 2'-F modification, "m" is the 2'-O-methyl modification, "I" is the internal base, and "*" is the phosphorothioate backbone linkage.

[0102] In any of the embodiments of antisense and siRNA molecules described herein, the molecules may contain one, two, or three additional nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends. In some embodiments, the antisense and siRNA molecules contain one, two, or three additional nucleotides at the 5' end. In some embodiments, the antisense and siRNA molecules contain one, two, or three additional nucleotides at the 3' end. In some embodiments, the antisense and siRNA molecules contain one, two, or three additional nucleotides at both the 5' and 3' ends.

[0103] In any of the embodiments of antisense and siRNA molecules described herein, the molecules can comprise substantially identical sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 80% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 85% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 90% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 95% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 98% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense and siRNA molecules have at least 99% homology to the nucleotide sequences disclosed herein.

[0104] In any of the embodiments described herein, the inhibitory nucleic acid molecule may be administered, for example, as a 1-2 hour intravenous infusion or subcutaneous injection. In any of the embodiments described herein, the inhibitory nucleic acid molecule may be administered at a dose of about 50 mg to about 900 mg, about 100 mg to about 800 mg, about 150 mg to about 700 mg, or about 175 to about 640 mg (mg / kg to mg / m2 based on an assumed 70 kg body weight and a mg / kg dose multiplier value for humans of 37). 2 Based on conversion to dose levels, 2.5 to 9.14 mg / kg, 92.5 to 338 mg / m 2 ) may be administered at dosage levels ranging from 0.1 to 100 mg / kg.

[0105] The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (e.g., cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.

[0106] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, etc.

[0107] In some embodiments, the method further comprises detecting the presence or absence of an ANGPTL3 variant nucleic acid molecule and detecting the presence or absence of an SLC5A2 variant nucleic acid molecule in a biological sample from the subject. The presence or absence of either an ANGPTL3 variant nucleic acid molecule or an SLC5A2 variant nucleic acid molecule can be detected herein. In some embodiments, the method further comprises administering a renal disease therapeutic agent to a subject that i) is heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and ii) is heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule.

[0108] The present disclosure also provides compositions comprising a combination of an ANGPTL3 inhibitor and an SLC5A2 inhibitor with a second therapeutic agent, such as (1) a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, or pravastatin; (2) an inhibitor of cholesterol uptake and / or bile acid reabsorption; (3) niacin, which increases lipoprotein catabolism; or (4) a fibrinogen activator, which reduces low-density lipoprotein (LDL) levels, improves high-density lipoprotein (HDL) and TG levels, and reduces the number of non-fatal heart attacks. (5) activators of LXR transcription factors that play a role in cholesterol removal, such as 22-hydroxycholesterol, or fixed combinations such as ezetimibe and simvastatin; statins with bile-binding compounds (e.g., cholestyramine, colestipol, colesveram), fixed combinations of niacin and statins (e.g., niacin and lovastatin); or combinations with other lipid-lowering drugs, such as omega-3 fatty acid ethyl esters (e.g., Omacor). Additionally, the second therapeutic agent may be one or more other inhibitors of ANGPTL3, as well as inhibitors of other molecules involved in lipid metabolism, particularly cholesterol and / or triglyceride homeostasis, such as ANGPTL4, ANGPTL5, ANGPTL6, and proprotein convertase subtilisin / kexin type 9 (PCSK9). Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity.

[0109] The present disclosure also provides methods of treating a subject with a renal disease therapeutic agent. In some embodiments, the subject has renal disease. In some embodiments, the subject is at risk of developing renal disease. The method includes administering a renal disease therapeutic agent to the subject. The method includes determining whether the subject has an ANGPTL3 variant nucleic acid molecule and whether the subject has an SLC5A2 variant nucleic acid molecule. The determination can be performed by obtaining or having obtained a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule. The method includes administering or continuing to administer a renal disease therapeutic agent to a subject that is homozygous for both the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. The method includes administering or continuing to administer a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who: i) is heterozygous for both an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule; ii) is heterozygous for one of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule and is a reference for the other of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule; or iii) is an ANGPTL3 reference and an SLC5A2 reference. The presence of a genotype having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule indicates that the subject has a reduced risk of developing kidney disease.

[0110] In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is diabetic kidney disease. In some embodiments, the kidney disease is kidney stones. In some embodiments, the kidney disease is chronic glomerulonephritis. In some embodiments, the kidney disease is nephronophthisis. In some embodiments, the kidney disease is chronic interstitial nephritis. In some embodiments, the kidney disease is nephrosclerosis.

[0111] In the case of a subject who has been genotyped or determined to be either i) heterozygous for an ANGPTL3 reference or an ANGPTL3 variant nucleic acid molecule, and ii) heterozygous for an SLC5A2 reference or an SLC5A2 variant nucleic acid molecule, such subject may be administered an ANGPTL3 inhibitor and an SLC5A2 inhibitor as described herein.

[0112] In some embodiments, the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide.

[0113] Detecting the presence or absence of ANGPTL3 variant nucleic acid molecules and SLC5A2 variant nucleic acid molecules in a biological sample from a subject and / or determining whether a subject has ANGPTL3 variant nucleic acid molecules and SLC5A2 variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules can be present in cells obtained from the subject.

[0114] In some embodiments, the subject is heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule, and the subject is administered or continues to be administered a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor, which can be any of the inhibitors described herein or any combination thereof.

[0115] In some embodiments, the treatment or prevention method further comprises detecting the presence or absence of an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide in a biological sample from the subject. In some embodiments, if the subject does not have an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide, the subject is administered a renal disease therapeutic agent and an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

[0116] The present disclosure also provides a method of treating a subject with a renal disease therapeutic agent, wherein the subject has renal disease or is at risk of developing renal disease. In some embodiments, the method includes obtaining or having obtained a biological sample from the subject and determining whether the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide by performing or having performed an assay on the biological sample to determine whether the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide. If the subject does not have an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide, a renal disease therapeutic agent is or continues to be administered to the subject, and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor are administered to the subject. The presence of an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide indicates that the subject has a reduced risk of developing renal disease. In some embodiments, the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide. In some embodiments, the subject does not have a NGPTL3 variant polypeptide and a SLC5A2 variant polypeptide.

[0117] Detecting the presence or absence of ANGPTL3 variant polypeptides and SLC5A2 variant polypeptides in a biological sample from a subject and / or determining whether a subject has ANGPTL3 variant polypeptides and SLC5A2 variant polypeptides can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptides can be present in cells obtained from the subject.

[0118] Examples of kidney disease therapeutic agents for treating chronic kidney disease and / or diabetic kidney disease include, but are not limited to, erythropoietin, diuretics (e.g., furosemide, bumetanide, ethacrynic acid, metolazone, and hydrochlorothiazide), blood pressure medications, phosphate binders, sodium bicarbonate, cholesterol medications, and gliflozin, or any combination thereof.

[0119] Examples of kidney disease therapeutic agents that treat or inhibit kidney stones include, but are not limited to, potassium citrate, diuretics (e.g., furosemide, bumetanide, ethacrynic acid, metolazone, and hydrochlorothiazide), allopurinol, acetohydroxamic acid, tamsulosin, nifedipine, d-penicillamine, tiopronin, and mercaptopropionylglycine, or any combination thereof.

[0120] Examples of kidney disease therapeutic agents that treat or inhibit chronic glomerulonephritis include, but are not limited to, angiotensin-converting enzyme (ACE) inhibitors (e.g., lisinopril, enalapril, captopril, benazepril, fosinopril, and quinapril), diuretics (e.g., furosemide, bumetanide, ethacrynic acid, metolazone, and hydrochlorothiazide), calcium channel blockers (e.g., amlodipine, nifedipine, felodipine, isradipine, verapamil, and diltiazem). azem), beta-adrenergic blocking agents (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), alpha-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), corticosteroids (e.g., prednisone), and immunosuppressants (e.g., cyclosphosphamide), or any combination thereof.

[0121] Examples of kidney disease therapeutic agents that treat or inhibit nephronophthisis include, but are not limited to, erythropoietin and blood pressure medication, or any combination thereof.

[0122] Examples of renal disease therapeutic agents that treat or inhibit chronic interstitial nephritis include, but are not limited to, corticosteroids, erythropoietin, blood pressure medications, statins, and chelating agents (e.g., succinimer and edetate calcium disodium), or combinations thereof.

[0123] Examples of renal disease therapeutic agents that treat or inhibit nephrosclerosis include, but are not limited to, diuretics (e.g., furosemide, bumetanide, ethacrynic acid, metolazone, and hydrochlorothiazide), ACE inhibitors (e.g., lisinopril, enalapril, captopril, benazepril, fosinopril, and quinapril), ARBs (e.g., losartan and valsartan), calcium channel blockers (e.g., amlodipine, nifedipine, felodipine, isradipine, verapamil, and diltiazem), beta- These include adrenergic blocking agents (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), alpha-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), renin inhibitors (e.g., aliskiren), vasodilators (e.g., minoxidil and hydralazine), and alpha-1 blockers (e.g., doxazosin), or any combination thereof.

[0124] In some embodiments, the dose of a renal disease therapeutic agent can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule compared to a subject who is homozygous for an ANGPTL3 variant nucleic acid molecule and homozygous for an ANGPTL3 variant nucleic acid molecule. In some embodiments, the dose of a renal disease therapeutic agent can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, a subject who is heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule may be administered less frequently compared to a subject who is homozygous for an ANGPTL3 variant nucleic acid molecule and homozygous for an ANGPTL3 variant nucleic acid molecule.

[0125] The administration of the renal disease therapeutic agent, ANGPTL3 inhibitor, and / or SLC5A2 inhibitor can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months.The repeated administration can be the same dose or different doses.The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more.For example, according to a certain dosage regimen, the subject can receive therapy for a long period of time, for example, six months, one year, or more.

[0126] Administration of the renal disease therapeutic agent, ANGPTL3 inhibitor, and / or SLC5A2 inhibitor can be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single administration dose). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0127] The terms "treat," "treating," and "treatment," as well as "prevent," "preventing," and "prevention," as used herein, refer to eliciting a desired biological response (such as a therapeutic effect and a prophylactic effect, respectively). In some embodiments, a therapeutic effect includes one or more of: a reduction / reduction in kidney disease, a reduction / reduction in the severity of kidney disease (such as, for example, reducing or inhibiting the onset of kidney disease), a reduction / reduction in symptoms and kidney disease-related effects, a delay in the onset of symptoms and kidney disease-related effects, a reduction in the severity of symptoms of kidney disease-related effects, a reduction in the number of symptoms and kidney disease-related effects, a reduction in the latency period of symptoms and kidney disease-related effects, an improvement in symptoms and kidney disease-related effects, a reduction in secondary symptoms, a reduction in secondary infections, prevention of relapse to kidney disease, a reduction in the number or frequency of recurrent episodes, an increase in the latency period between symptomatic episodes, an increase in the time to sustained progression, a more rapid recovery, or an increase in the effectiveness of or a decrease in resistance to alternative therapeutic agents, and / or an increase in the survival time of an affected host animal after administration of the agent or a composition comprising the agent. A prophylactic effect can include complete or partial avoidance / inhibition, or delay (e.g., complete or partial avoidance / inhibition, or delay, etc.) of the onset / progression of kidney disease following administration of a treatment protocol, and prolongation of survival of an affected host animal. Treatment of kidney disease encompasses treatment of a subject already diagnosed as having any form of kidney disease at any clinical stage or symptom, delaying the onset or progression or worsening or deterioration of symptoms or signs of kidney disease, and / or preventing and / or reducing the severity of kidney disease.

[0128] The present disclosure also provides a method for identifying a subject at high risk of developing kidney disease. The method further includes determining or having determined the presence or absence of an ANGPTL3 variant nucleic acid molecule and the presence or absence of an SLC5A2 variant nucleic acid molecule in a biological sample obtained from the subject. If the subject is ANGPTL3 reference and SLC5A2 reference, the subject has an increased risk of developing kidney disease. If the subject is heterozygous or homozygous for the ANGPTL3 variant nucleic acid molecule and heterozygous or homozygous for the SLC5A2 variant nucleic acid molecule, or if the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and the reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, the subject has a reduced risk of developing kidney disease.

[0129] In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is diabetic kidney disease. In some embodiments, the kidney disease is kidney stones. In some embodiments, the kidney disease is chronic glomerulonephritis. In some embodiments, the kidney disease is nephronophthisis. In some embodiments, the kidney disease is chronic interstitial nephritis. In some embodiments, the kidney disease is nephrosclerosis.

[0130] In some embodiments, the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide. In some embodiments, the ANGPTL3 variant nucleic acid molecule is any of the ANGPTL3 variant nucleic acid molecules described herein.

[0131] In some embodiments, the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is any of the SLC5A2 variant nucleic acid molecules described herein.

[0132] Having a single copy of an ANGPTL3 variant nucleic acid molecule and / or a single copy of an SLC5A2 variant nucleic acid molecule provides a subject with greater protection from developing kidney disease than having no copies of an ANGPTL3 variant nucleic acid molecule and no copies of an SLC5A2 variant nucleic acid molecule. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of an ANGPTL3 variant nucleic acid molecule and a single copy of an SLC5A2 variant nucleic acid molecule (i.e., heterozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and references the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule) will protect a subject from developing kidney disease, and that two copies of an ANGPTL3 variant nucleic acid molecule and two copies of the SLC5A2 variant nucleic acid molecule (i.e., homozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule) may be more protective of a subject from developing kidney disease compared to a subject having a single copy of each. Thus, in some embodiments, a single copy of an ANGPTL3 variant nucleic acid molecule and a single copy of an SLC5A2 variant nucleic acid molecule may not completely protect a subject from developing kidney disease, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, it is possible that there are additional factors or molecules involved in the development of kidney disease that are still present in a subject having a single copy of an ANGPTL3 variant nucleic acid molecule and a single copy of an SLC5A2 variant nucleic acid molecule, resulting in less than complete protection from developing kidney disease.

[0133] Determining whether a subject has an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule in a biological sample from the subject and / or determining whether a subject has an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules can be present in cells obtained from the subject.

[0134] In some embodiments, the method further comprises administering a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who is i) heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule and ii) heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule. In some embodiments, the ANGPTL3 inhibitor and the SLC5A2 inhibitor comprise any of the inhibitors described herein. In some embodiments, the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to the ANGPTL3 nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to ERAP2 mRNA. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the ANGPTL3 inhibitor comprises a small molecule. In some embodiments, the ANGPTL3 inhibitor comprises an antibody. In some embodiments, the antibody comprises evinacumab. In some embodiments, the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an SLC5A2 nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to ERAP2 mRNA. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof.

[0135] The present disclosure also provides methods for determining the total burden, or polygenic risk score (PRS), of a subject with two or more ANGPTL3 and SLC5A2 variant nucleic acid molecules and / or two or more ANGPTL3 and SLC5A2 variant polypeptides associated with a reduced risk of developing kidney disease. The total burden is the sum of two or more genetic variants that are viable in association studies with kidney disease. In some embodiments, the subject is homozygous for one or more ANGPTL3 and SLC5A2 variant nucleic acid molecules associated with a reduced risk of developing kidney disease. In some embodiments, the subject is heterozygous for one or more ANGPTL3 and SLC5A2 variant nucleic acid molecules associated with a reduced risk of developing kidney disease. In some embodiments, the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, and is reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. When the subject's total burden is low, the subject has an increased risk of developing kidney disease, and the subject is administered or continues to be administered with a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor.When the subject's total burden is high, the subject has a decreased risk of developing kidney disease, and the subject is administered or continues to be administered with a kidney disease therapeutic agent.The higher the total burden, the lower the risk of developing kidney disease.

[0136] In some embodiments, the total burden of a subject with any two or more ANGPTL3 and SLC5A2 variant nucleic acid molecules represents the weighted sum of any plurality of ANGPTL3 and SLC5A2 variant nucleic acid molecules. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants present in or around the ANGPTL3 and SLC5A2 genes (up to 10 Mb), where the genetic burden is the number of alleles multiplied by the estimated association with kidney disease or associated outcome for each allele (e.g., a weighted polygenic burden score). In some embodiments, if the subject has a total burden higher than the desired threshold score, the subject is at a decreased risk of developing kidney disease, hi some embodiments, if the subject has a total burden lower than the desired threshold score, the subject is at an increased risk of developing kidney disease.

[0137] In some embodiments, the total burden may be divided into quintiles, for example, the top quintile, the second quintile, the middle quintile, the fourth quintile, and the bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with higher total burdens have the highest weighted total burdens, including, but not limited to, the top 10%, top 20%, top 30%, top 40%, or top 50% total burdens from the subject population. In some embodiments, the genetic variants include genetic variants with association with kidney disease in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants has a p-value of about 10 -2 Below, about 10 -3 Below, about 10 -4 Below, about 10 -5 Below, about 10 -6 Below, about 10 -7 Below, about 10 -8 Below, about 10 -9 Below, about 10 -10 Below, about 10 -11 Below, about 10 -12 Below, about 10 -13 Below, about 10 -14 Less than or equal to 10 -15 In some embodiments, the identified genetic variants include those with an association with kidney disease that have a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants having an association with kidney disease in high-risk subjects with an odds ratio (OR) of about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater for the top 20% of the distribution, or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater for the remainder of the reference population. In some embodiments, the odds ratio (OR) can be in the range of about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or greater than 7.0. In some embodiments, a high-risk subject has a total burden in the bottom decile, quintile, or tertile of the reference population. The total burden threshold can be determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0138] The present disclosure also provides methods for detecting the presence or absence of ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules generated from mRNA molecules) in a biological sample from a subject. It is understood that gene sequences within a population and mRNA molecules encoded by such genes may vary due to polymorphisms, such as single nucleotide polymorphisms.

[0139] A biological sample can be derived from any cell, tissue, or biological fluid from a subject. The biological sample can include any clinically relevant tissue, such as a bone marrow sample, tumor biopsy, fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample can be processed differently depending on the assay used. For example, when detecting any ANGPTL3 and SLC5A2 variant nucleic acid molecules, pretreatment designed to isolate or enrich the biological sample for genomic DNA can be employed. For this purpose, various techniques can be used. When detecting the level of any ANGPTL3 and SLC5A2 variant nucleic acid molecules, various techniques can be used to enrich the biological sample for mRNA molecules. Various methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0140] In some embodiments, detecting ANGPTL3 and SLC5A2 variant nucleic acid molecules in a subject comprises performing sequence analysis on a biological sample obtained from the subject to determine whether ANGPTL3 and SLC5A2 genomic nucleic acid molecules in the biological sample, and / or ANGPTL3 and SLC5A2 mRNA molecules in the biological sample, and / or ANGPTL3 and SLC5A2 cDNA molecules generated from the mRNA molecules in the biological sample are present in the sample. In some embodiments, the method detects ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules, or mRNA molecules generated therefrom, or cDNA molecules generated from the mRNA molecules, that comprise any of the genetic mutations described herein.

[0141] In some embodiments, methods for detecting the presence or absence of ANGPTL3 and SLC5A2 variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules generated from mRNA molecules, etc.) in a subject include performing an assay on a biological sample obtained from the subject, which assay determines whether a nucleic acid molecule in the biological sample contains a particular nucleotide sequence.

[0142] In some embodiments, the biological sample comprises cells or cell lysates. Such a method can further comprise, for example, obtaining a biological sample comprising ANGPTL3 and SLC5A2 genomic nucleic acid molecules or mRNA molecules from a subject, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such an assay can comprise, for example, determining the identity of these positions of specific ANGPTL3 and SLC5A2 nucleic acid molecules. In some embodiments, the method is an in vitro method.

[0143] In some embodiments, the determining, detecting, or sequence analyzing step comprises sequencing at least a portion of the nucleotide sequence of an ANGPTL3 and SLC5A2 genomic nucleic acid molecule, an ANGPTL3 and SLC5A2 mRNA molecule, or an ANGPTL3 and SLC5A2 cDNA molecule in a biological sample containing a genetic variation compared to a corresponding ANGPTL3 and SLC5A2 reference molecule. In some embodiments, the sequenced portion contains one or more variations that cause or are predicted to cause a loss of function (partial or complete).

[0144] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the ANGPTL3 and SLC5A2 genomic nucleic acid molecules are analyzed. In some embodiments, only the ANGPTL3 and SLC5A2 mRNAs are analyzed. In some embodiments, only the ANGPTL3 and SLC5A2 cDNAs derived from the ANGPTL3 and SLC5A2 mRNAs are analyzed.

[0145] Mutation-specific polymerase chain reaction techniques can be used to detect mutations (such as SNPs in nucleic acid sequences) because variation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0146] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0147] In some embodiments, the assay involves contacting the biological sample under stringent conditions with a primer or probe, such as a mutation-specific primer or a mutation-specific probe, that specifically hybridizes to ANGPTL3 and SLC5A2 variant genomic sequences, variant mRNA sequences, or variant cDNA sequences, but not to the corresponding ANGPTL3 and SLC5A2 reference sequences, and determining whether hybridization occurs.

[0148] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of an ANGPTL3 and SLC5A2 nucleic acid molecule encoding an ANGPTL3 and SLC5A2 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support containing a mutation-specific probe; and d) detecting the detectable label.

[0149] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0150] In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to target nucleotide sequences and specifically detect and / or identify polynucleotides comprising ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the practitioner to achieve this result. The nucleotide length may be any length sufficient for use in an optimal detection method, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers may have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, or probes that differ from the target nucleotide sequence but retain the ability to specifically detect and / or distinguish the target nucleotide sequence may be designed using conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0151] Exemplary nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0152] In hybridization techniques, stringent conditions can be used to ensure that a probe or primer specifically hybridizes to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence at a detectably higher degree (at least 2-fold above background, at least 3-fold above background, at least 4-fold above background, or more, including more than 10-fold above background) than other non-target sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at a detectably higher degree (at least 2-fold) than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at a detectably higher degree (at least 3-fold) than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at a detectably higher degree (at least 4-fold) than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably higher degree (more than 10-fold over background) than to other nucleotide sequences. Stringent conditions are sequence-dependent and will vary depending on the circumstances.

[0153] Suitable stringent conditions for promoting DNA hybridization, such as 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by a 2× SSC wash at 50° C., are known or can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection are those in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ions, typically about 0.01 to 1.0 M Na + The stringent conditions are those with a high ionic concentration (or other salts) and a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by adding a destabilizing agent such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0154] In some embodiments, such isolated nucleic acid molecules have a nucleotide sequence of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 to about 35 nucleotides.

[0155] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to ANGPTL3 and SLC5A2 variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). Such nucleic acid molecules can be used, for example, as probes, primers, variant-specific probes, or variant-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0156] In some embodiments, the isolated nucleic acid molecule hybridizes with at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an ANGPTL3 or SLC5A2 variant nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0157] In some embodiments, the mutation-specific probe and mutation-specific primer comprise DNA. In some embodiments, the variation-specific probe and variation-specific primer comprise RNA.

[0158] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein, or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0159] In some embodiments, primers (including mutation-specific primers) can be used in second-generation or high-throughput sequencing. In some instances, primers, including mutation-specific primers, can be modified. In particular, primers can contain various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is generally performed using a paired-end tag library in which each molecule of DNA template is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain 5'-biotin tags for immobilization of DNA libraries on streptavidin-coated beads.

[0160] The probes and primers described herein can be used to detect nucleotide variations in any of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any ANGPTL3 and SLC5A2 variant nucleic acid molecule, or fragment thereof.

[0161] In the context of the present disclosure, "specifically hybridize" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer, etc.) does not hybridize to a nucleic acid sequence encoding an ANGPTL3 and SLC5A2 reference genomic nucleic acid molecule, an ANGPTL3 and SLC5A2 reference mRNA molecule, and / or an ANGPTL3 and SLC5A2 reference cDNA molecule.

[0162] In some embodiments, the probe (e.g., mutation-specific probe, etc.) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0163] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are bound. A solid support is a solid-state substrate or support to which molecules such as any of the probes disclosed herein can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different probes are bound in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multi-well glass slide can be used, usually containing one array per well.

[0164] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be derived from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be derived from humans or orthologs from other organisms, such as non-human mammals, rodents, mice, or rats. It is understood that gene sequences within a population may differ due to polymorphisms, such as single nucleotide polymorphisms.

[0165] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can function as effectors, inhibitors, regulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have novel activities independent of any other molecules.

[0166] The isolated nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, such as vectors or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be within a vector or an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent, metal-containing, or enzymes, resulting in enzyme-dependent secondary signal generation. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a conjugated molecule so that when the conjugated molecule is subsequently added with a substrate, it can be used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and tested with a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose-binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent, and chemiluminescent substrates, as well as other labels.

[0167] Percent identity (or percent complementarity) between specific regions of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be routinely determined using the BLAST program (basic alignment search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489), using default settings. Appl. Math., 1981, 2, 482-489) using the default settings of the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.). As used herein, when referring to percent sequence identity, higher percentages of sequence identity are preferred over lower ones.

[0168] The present disclosure also provides a renal disease therapeutic agent for use in treating or preventing renal disease in a subject having an ANGPTL3 or SLC5A2 variant nucleic acid molecule. Any of the renal disease therapeutic agents described herein can be used herein. Any of the ANGPTL3 or SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the PRS comprises an SCN11A variant genomic nucleic acid molecule containing any of the genetic mutations described herein, or is an mRNA molecule generated therefrom, or is a cDNA molecule generated from an mRNA molecule.

[0169] The present disclosure also provides use of a renal disease therapeutic agent for use in preparing a medicament for treating or preventing renal disease in a subject having an ANGPTL3 or SLC5A2 variant nucleic acid molecule. Any of the renal disease therapeutic agents described herein can be used herein. Any of the ANGPTL3 or SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the PRS comprises an SCN11A variant genomic nucleic acid molecule containing any of the genetic mutations described herein, or is an mRNA molecule generated therefrom, or is a cDNA molecule generated from the mRNA molecule.

[0170] The present disclosure also provides an ANGPTL3 and SLC5A2 inhibitor for use in treating or preventing kidney disease in a subject who is heterozygous for a reference ANGPTL3 and SLC5A2 or a variant ANGPTL3 and SLC5A2 nucleic acid molecule, or who is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. Any of the ANGPTL3 and SLC5A2 inhibitors described herein can be used herein. Any of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the PRS comprises an SCN11A variant genomic nucleic acid molecule containing any of the genetic mutations described herein, or is an mRNA molecule generated therefrom, or is a cDNA molecule generated from the mRNA molecule.

[0171] The present disclosure also provides an ANGPTL3 and SLC5A2 inhibitor in the preparation of a medicament for treating or preventing kidney disease in a subject who is heterozygous for a reference ANGPTL3 and SLC5A2 or a variant ANGPTL3 and SLC5A2 nucleic acid molecule, or who is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. Any of the ANGPTL3 and SLC5A2 inhibitors described herein can be used herein. Any of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the PRS comprises an SCN11A variant genomic nucleic acid molecule containing any of the genetic mutations described herein, or is an mRNA molecule generated therefrom, or is a cDNA molecule generated from the mRNA molecule.

[0172] In some embodiments, the ANGPTL3 and SLC5A2 inhibitor and the renal disease therapeutic agent are disposed within a pharmaceutical composition. In some embodiments, the ANGPTL3 and SLC5A2 inhibitor are disposed within a first pharmaceutical composition, and the renal disease therapeutic agent is disposed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition. Alternatively, the ANGPTL3 inhibitor, the SLC5A2 inhibitor, and the renal disease therapeutic agent are disposed within separate pharmaceutical compositions.

[0173] All patent documents, websites, other publications, accession numbers, and the like, cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where the version of a sequence associated with an accession number varies over time, the version associated with that accession number as of the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date for which the accession number is provided or the filing date of a priority application, if applicable. Similarly, where different versions of publications, websites, etc. are published at different times, the version last published as of the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. While the present disclosure has been described in some detail by illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0174] The following examples are provided to more fully describe the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those of ordinary skill in the art with a disclosure and description of how the compounds, compositions, articles, devices, and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and variation should be expected. Unless otherwise indicated, parts are parts by weight, temperature is in ° C. or is ambient temperature, and pressure is at or near atmospheric. [Example]

[0175] Example 1: Loss of function of ANGPTL3 is associated with improved renal function, independent of SLC5A2 To determine whether the effects of ANGPTL3 on chronic kidney disease are influenced by SGLT2 function, we performed exome sequencing on up to 677,048 participants across five studies to identify rare coding variants. We estimated the association between the burden of identified rare loss-of-function and missense variants in ANGPTL3 and eGFR, with or without adjustment for the burden of rare loss-of-function and missense variants in SLC5A2, the gene encoding SGLT2. eGFR is a widely used biomarker of kidney function, with higher levels indicating better function.

[0176] In our analysis, the burden of rare (alternative allele frequency (AAF) <0.1%) predicted loss-of-function (pLOF) or missense genetic variants in ANGPTL3 was strongly associated with increased eGFR (Table 1). Importantly, the burden of pLOF or missense genetic variants in ANGPTL3 had a similar effect on increased eGFR after adjusting for rare loss-of-function and missense variants in SLC5A2 (AAF <0.1%), indicating that the two genes act independently of each other to increase eGFR. [Table 1]

[0177] Similar results with an increase were observed comparing associations of rare ANGPTL3 pLOF variants alone (AAF<1%, excluding missense variants) with or without adjustment for SLC5A2 pLOF variants (see Table 2), indicating that associations of rare pLOF+missense variants reflect loss of function in ANGPTL3 independent of loss of function effects in SLC5A2. [Table 2]

[0178] Example 2: Loss of function of SLC5A2 is associated with improved renal function, independent of ANGPTL3 We next compared the association of rare coding variants in SLC5A2 with increased eGFR with and without adjustment for ANGPTL3 rare coding variants. The burden of pLOF or missense genetic variants in SLC5A2 (AAF < 0.1%) had a similar effect on increased eGFR after adjusting for rare loss-of-function and missense variants in ANGPTL3 (AAF < 0.1%) compared with no adjustment, indicating that the two genes act independently of each other to increase eGFR (see Table 3). [Table 3]

[0179] Similar results with an increase were observed comparing associations of rare SLC5A2 pLOF variants alone (AAF<1%, excluding missense variants) with or without adjustment for ANGPTL3 pLOF variants (see Table 4), indicating that associations of rare pLOF+missense variants reflect loss of function in SLC5A2 independent of loss of function effects in ANGPTL3. [Table 4]

[0180] Example 3: Rare coding variants in ANGPTL3 and SLC5A2 are associated with improved renal function, independent of other genes To formally test whether the effects of NGPTL3 and SLC5A2 rare-coding variants on eGFR were independent of each other, an interaction analysis was performed. The main effects of ANGPTL3 (i.e., the effect of ANGPTL3 variants in individuals with a homozygous reference genotype for SLC5A2 variants) and SLC5A2 (i.e., the effect of SLC5A2 variants in individuals with a homozygous reference genotype for ANGPTL3 variants) on eGFR were similar to the overall effects of ANGPTL3 rare-coding variants (see Table 1) or SLC5A2 rare-coding variants (see Table 3), respectively (see Table 5). The interaction terms testing whether the effect of ANGPTL3 variants differed in SLC5A2 homozygous references compared to SLC5A2 heterozygous variant carriers and whether the effect of SLC5A2 variants differed in ANGPTL3 homozygous references compared to ANGPTL3 heterozygous variant carriers were not significant (see Table 5). This indicates that the effect of ANGPTL3 is independent of SLC5A2 genotype status, and that the effect of SLC5A2 is independent of ANGPTL3 genotype status, and therefore there is no evidence of deviation from additivity for the association of these two genotypes. [Table 5]

[0181] The association between ANGPTL3, SLC5A2, and eGFR was driven by multiple rare pLOF or missense variants in the ANGPTL3 and SLC5A2 genes (see Table 6). [Table 6-1] [Table 6-2] [Table 6-3] Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24 Table 6-25 Table 6-26 Table 6-27 Table 6-28 Table 6-29 Table 6-30 Table 6-31 Table 6-32 Table 6-33 Table 6-34 Table 6-35 Table 6-36 Table 6-37 Table 6-38 Table 6-39 Table 6-40 Table 6-41 Table 6-42 Table 6-43 Table 6-44 Table 6-45 Table 6-46 Table 6-47 Table 6-48 Table 6-49 Table 6-50 Table 6-51 Table 6-52 Table 6-53 Table 6-54 Table 6-55 Table 6-56 Table 6-57 Table 6-58 Table 6-59 Table 6-60 Table 6-61 Table 6-62 Table 6-63 Table 6-64 Table 6-65 Table 6-66 Table 6-67 Table 6-68 Table 6-69 Table 6-70 Table 6-71 Table 6-72 Table 6-73 Table 6-74 Table 6-75 Table 6-76 Table 6-77 Table 6-78 Table 6-79 Table 6-80 Table 6-81 Table 6-82 Table 6-83 Table 6-84 Table 6-85 Table 6-86 [Table 6-87]

[0182] Example 4: General Methods Participating cohort UKB is a population-based cohort study of individuals aged 40-69 years recruited through 22 UK testing centers between 2006 and 2010 (Sudlow et al., PLoS Med, 2015, 12, e1001779). A total of 431,835 participants from UKB with available whole-exome sequencing and phenotype data were included. The MyCode Community Health Initiative cohort from Geisinger Health System (GHS) (Carey et al., Genet. Med, 2016, 18, 906-913) is a health-system-based cohort of patients from Central and Eastern Pennsylvania (USA) recruited between 2007 and 2019. A total of 156,846 participants from GHS with available whole-exome sequencing and phenotype data were included. The Mount Sinai BioMe Precision Medicine Cohort (SINAI) (Gottesman et al., Genet. Med., 2013, 15, 761-771) is a clinical care cohort linked to electronic health records of 25,839 individuals with available whole-exome sequencing and phenotypes. The University of Pennsylvania Medicine BioBank (UPENN-PMBB) is a health-system-based cohort based at the Pennsylvania BioBank (Park et al., Nat. Med., 2021, 27, 66-72). The Malmo Diet and Cancer Study (MDCS) is a cohort study based in Malmö, Sweden (Berglund et al., J. Intern. Med., 1993, 233, 45-51). The MCPS is a population-based prospective study based in Mexico (Tapia-Conyer et al., Int. J. Epidemiol., 2006 35, 243-49).

[0183] Phenotype definition eGFR was calculated from laboratory measurements of creatinine extracted from the electronic health records (EHRs) of participants from the GHS, SINAI, and UPENN-PMBB. The median was calculated for all participants with two or more measurements. In the UKB, eGFR was calculated from creatinine measured on a Beckman Coulter AU5800 clinical chemistry analyzer, and cystatin was measured by immunoturbidimetry on a Siemens Advia 1800 clinical chemistry analyzer. Both creatinine and cystatin were measured at the study's baseline visit. In the MCPS, eGFR was calculated from creatinine measured by nucleic acid magnetic resonance spectroscopy and converted to clinical units as previously described (Aguilar-Ramirez et al., J. Clin. Endo. Met., 2021, 106, 2828-2839). eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration formula for creatinine and cystatin (UKB) or creatinine (all other cohorts). Prior to genetic association analysis, eGFR values ​​were transformed by the inverse standard normal function, applied within each ancestry group and separately for men and women.

[0184] Genotype data High-coverage whole-exome sequencing was performed as previously described (Science, 2021, 373:abf8683 and Nature, 2020, 586:749-756) and summarized below. NimbleGen probes (VCRome, for a portion of the GHS cohort) or a modified version of the xGen design available from Integrated DNA Technologies (IDT, for the remainder of the GHS and other cohorts) were used for targeted exome capture. To facilitate multiplexed exome capture and sequencing, a unique 6-base pair (bp) barcode (VCRome) or 10-bp barcode (IDT) was added to each DNA fragment during library preparation. Equal amounts of sample were pooled prior to exome capture. Sequencing was performed using 75-bp paired-end reads on an Illumina v4 HiSeq 2500 instrument (for a portion of the GHS cohort) or a NovaSeq instrument (for the remainder of the GHS and other cohorts). Sequencing had sufficient coverage depth (i.e., the number of sequence reads covering each nucleotide in the target region of the genome) to provide >20x coverage across 85% of the target bases in 96% of the VCRome samples and >20x coverage across 90% of the target bases in 99% of the IDT samples. Data processing steps included sample demultiplexing using Illumina software, alignment to the GRCh38 human genome reference sequence, including generation of binary alignment and mapping files (BAM), and processing of the BAM files (e.g., marking duplicate reads and other read mapping evaluations). Variant calling was performed using the GLNexus system (DOI: 10.1101 / 343970). Variant mapping and annotation were based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. Next, snpEff predictions relating to protein-coding transcripts with annotated start and stop points were combined into a single functional impact prediction by selecting the most deleterious functional effect class for each gene.The hierarchy of these annotations (from most deleterious to least deleterious) was frameshift, stop-gain, stop-loss, splice acceptor, splice donor, stop-lost, in-frame indel, missense, and other. Predicted LOF gene variants included a) insertions or deletions resulting in frameshifts, b) insertions, deletions, or single-nucleotide variants resulting in the introduction of premature stop codons or loss of transcription start or stop sites, and c) donor or acceptor splice site variants. Missense variants were classified for their potential functional impact according to a number of in silico prediction algorithms that predicted deleteriousness using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9) and Polyphen2_HVAR (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), LRT (Chun et al., Genome Res., 2009, 19, 1553-61), and MutationTaster (Schwarz et al., Nat. Methods, 2010, 7, 575-6). For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determined inclusion into seven gene burden exposures: 1) pLOF variants with AAF < 1%, 2) pLOF or missense variants predicted to be deleterious by 5 of 5 algorithms with AAF < 1%, 3) pLOF or missense variants predicted to be deleterious by 5 of 5 algorithms with AAF < 0.1%, 4) pLOF or missense variants predicted to be deleterious by at least 1 of 5 algorithms with AAF < 1%, 5) pLOF or missense variants predicted to be deleterious by at least 1 of 5 algorithms with AAF < 0.1%, 6) pLOF or any missense with AAF < 1%, and 7) pLOF or any missense variant with AAF < 0.1%.

[0185] Association analysis of the genetic burden of rare pLOF mutations and missense mutations We used REGENIE (Mbatchou et al., Nat. Genetics, 2021, 53, 1097-1103) to test the association between the burden of rare predicted loss-of-function or missense variants in a given gene and eGFR by fitting a linear regression model adjusted for a polygenic score that approximates a genomic relationship matrix. Analyses were stratified by ancestry and included age, age, and sex. 2 , Gender, Age - Gender and age 2 The analyses were adjusted for a sex interaction term, experimental batch-related covariates, principal components derived from 10 common variants, and principal components derived from 20 rare variants. Results across cohorts for each variant-phenotype association were combined using fixed-effects inverse variance-weighted meta-analysis. For gene burden testing, all individuals were labeled as heterozygous if they had one or more eligible rare variants (as described above based on frequency and functional annotation) and as homozygous if they had any eligible variant in the homozygous state. This "composite genotype" was then used to test for association. The exposure burden with the strongest association with eGFR and the burden of pLOF variants with AAF < 1% were carried forward for further analysis.

[0186] To investigate whether the effect of ANGPTL3 gene burden on eGFR varies depending on SLC5A2 gene burden, or vice versa, we took two approaches. First, we adjusted for the second gene burden by including it as a covariate in a linear regression model between the first gene burden and eGFR. These results were compared with results from a linear regression model between the first gene burden and eGFR without adjusting for the second gene burden. Second, we performed a formal interaction analysis of eGFR using a linear regression model with ANGPTL3 gene burden, SLC5A2 gene burden, and an interaction term for ANGPTL3*SLC5A2 gene burden. The p-value of the interaction term allows us to assess whether the effect of ANGPTL3 gene burden significantly differs between different genotypes of SLC5A2 gene burden (and vice versa). Both analyses were performed as described in the previous paragraph, substituting a modified REGENIE polygenic score that excludes both chromosomes containing the ANGPTL3 and SLC5A2 genes.

[0187] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, GenBank accession numbers, etc.) is incorporated herein by reference in its entirety for all purposes.

Claims

1. An in vitro method for assessing a subject's risk of developing kidney disease, said method comprising: determining or having determined the presence or absence of an ANGPTL3 variant nucleic acid molecule in a biological sample obtained from the subject, and determining or having determined the presence or absence of an SLC5A2 variant nucleic acid molecule; ANGPTL3-reference and SLC5A2-reference in the subject is an indication that the subject is at increased risk for developing kidney disease; The method, wherein the subject being heterozygous or homozygous for the ANGPTL3 variant nucleic acid molecule and heterozygous or homozygous for the SLC5A2 variant nucleic acid molecule, or the subject being heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and reference for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, is an indication that the subject has a reduced risk of developing kidney disease.

2. 2. The method of claim 1, wherein the kidney disease is chronic kidney disease, diabetic kidney disease, chronic glomerulonephritis, nephronophthisis, chronic interstitial nephritis, or nephrosclerosis.

3. 3. The method of claim 1 or 2, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide.

4. 3. The method of claim 1 or 2, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide.

5. A composition for treating or preventing kidney disease in a subject having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule, the composition comprising a kidney disease therapeutic agent.

6. 6. The composition of claim 5, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide.

7. 6. The composition of claim 5, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide.

8. 1. A composition for use in treating or preventing renal disease in a subject i) who is heterozygous for an angiopoietin-like 3 (ANGPTL3) reference or ANGPTL3 variant nucleic acid molecule, and ii) who is heterozygous for a solute carrier family 5 member 2 (SLC5A2) reference or SLC5A2 variant nucleic acid molecule, the composition comprising an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

9. 9. The composition of claim 8, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated ANGPTL3 polypeptide.

10. 9. The composition of claim 8, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated SLC5A2 polypeptide.

11. 9. The composition of claim 8, wherein the ANGPTL3 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to an ANGPTL3 nucleic acid molecule.

12. The composition of claim 8 , wherein the ANGPTL3 inhibitor comprises a small molecule.

13. The composition of claim 8 , wherein the ANGPTL3 inhibitor comprises an antibody.

14. 14. The composition of claim 13, wherein the antibody comprises evinacumab.

15. 9. The composition of claim 8, wherein the SLC5A2 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to an SLC5A2 nucleic acid molecule.

16. 11. The composition of any one of claims 8 to 10, wherein the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof.

17. The composition described in any one of claims 5 to 7, wherein the renal disease is chronic renal disease, diabetic renal disease, chronic glomerulonephritis, nephronophthisis, chronic interstitial nephritis, or nephrosclerosis.

18. A composition described in any one of claims 8 to 15, wherein the renal disease is chronic renal disease, diabetic renal disease, chronic glomerulonephritis, nephronophthisis, chronic interstitial nephritis, or nephrosclerosis.