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

Abstract

The present disclosure provides methods of treating a subject having a kidney disease or preventing a subject from developing into a kidney disease by administering an angiopoietin-like 3 (ANGPTL3) inhibitor and a solute carrier family 5 member 2 (SLC5A2) inhibitor, and methods of identifying a subject having an increased risk of developing into a kidney disease.

Description

[0001] Reference to Sequence Listing

[0002] This application includes a Sequence Listing that has been submitted electronically as an XML file named 381203963SEQ, created on November 9, 2023, and having a size of 3,455,297 bytes. The Sequence Listing is hereby incorporated by reference. Technical Field

[0003] The present disclosure generally relates to treating subjects with kidney disease with angiopoietin-like 3 (ANGPTL3) inhibitors and solute carrier family 5 member 2 (SLC5A2) inhibitors, and methods of identifying subjects at increased risk of developing kidney disease. Background Art

[0004] In the United States, based on data from the National Health and Nutrition Examination Survey (NHANES) studies from 1999 to 2006, an estimated 11.1% (22.4 million) of adults 20 years of age or older had chronic kidney disease (CKD) stages 1 - 3. Additionally, 800,000 adults 20 years of age or older in the United States had CKD stage 4, and more than 300,000 had CKD stage 5 and were receiving hemodialysis. Analysis of NHANES data between 1988 - 1994 and 1999 - 2004 showed that the prevalence of CKD increased for each CKD stage, but the increase in prevalence was particularly notable for individuals classified as having CKD stage 3. The number of CKD stage 5 patients requiring dialysis was 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 diseases (e.g., glomerular diseases, tubulointerstitial diseases, obstruction, and polycystic kidney disease), in the vast majority of CKD patients, kidney damage is associated with other medical conditions such as diabetes and hypertension. In 2008, excluding ESRD patients, 48% of Medicare CKD patients had diabetes, 91% had hypertension, and 46% had atherosclerotic heart disease. Other risk factors for CKD include age, obesity, family history, and race. CKD is associated with many adverse health outcomes.

[0005] In most healthy individuals, a glomerular filtration rate (GFR) of 90 mL / min or higher (Stage 1) is normal. In this stage of CKD, symptoms are usually rare. For some patients (such as the elderly or infants), a GFR of 60 - 89 mL / min (Stage 2) may be normal if there is no kidney damage. If the GFR is between 60 - 89 mL / min and persists for three months or longer, along with kidney damage, it is a sign of early CKD. In this stage, symptoms are usually rare. A patient with a GFR between 30 - 59 mL / min (Stage 3) indicates moderate CKD and is more likely to develop anemia, early bone disease, or hypertension and may need to see a nephrologist. A GFR between 15 - 29 mL / min (Stage 4) indicates that the patient has severe CKD and may 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 has ESRD. In this stage, the kidneys have almost completely lost all ability to function effectively. They need dialysis or a kidney transplant to survive.

[0006] The ANGPTL3 gene encodes a member of a family of secreted proteins that play a role in angiogenesis. The encoded protein is mainly expressed in the liver and is further processed into a chain containing an N-terminal coiled-coil domain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain can be involved in angiogenesis. Mutations in this gene result in familial hypobetalipoproteinemia type 2.

[0007] The SLC5A2 gene encodes a low-affinity, high-capacity Na(+) / glucose cotransporter that is located in the early proximal tubule segment S1 and has a Na(+)-to-glucose coupling ratio of 1:1. It is the major mechanism for D-glucose reabsorption in the kidney. Summary of the Invention

[0008] The present disclosure provides methods of treating a subject having a kidney disease or at risk of developing a kidney disease, the methods comprising administering to the subject an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

[0009] The present disclosure also provides methods of treating a subject with a therapeutic agent for kidney disease by administering the therapeutic agent for kidney disease, wherein the subject has kidney disease or is at risk of developing kidney disease, the methods comprising: determining whether the subject has an ANGPTL3 variant nucleic acid molecule and whether the subject has an SLC5A2 variant nucleic acid molecule by the following steps: 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 continuing to administer the therapeutic agent for kidney disease to a subject who is homozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule; or administering or continuing to administer the therapeutic agent for kidney disease and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who is: i) heterozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, ii) heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and of a reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or iii) of an ANGPTL3 reference type and an SLC5A2 reference type; wherein the presence of a genotype comprising an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule indicates a reduced risk of the subject developing kidney disease.

[0010] The present disclosure also provides methods of identifying a subject at increased risk of developing kidney disease, the methods 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 in the biological sample; wherein: when the subject is of an ANGPTL3 reference type and an SLC5A2 reference type, then the subject has an increased risk of developing kidney disease; and when 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 when the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and of a reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, then the subject has a reduced risk of developing kidney disease.

[0011] The present disclosure also provides a therapeutic agent for kidney disease for use in treating or preventing kidney disease in a subject having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule.

[0012] The present disclosure also provides ANGPTL3 inhibitors and SLC5A2 inhibitors for use in treating or preventing kidney diseases in a subject who is: i) heterozygous for an ANGPTL3 reference or ANGPTL3 variant nucleic acid molecule, ii) heterozygous for an SLC5A2 reference or SLC5A2 variant nucleic acid molecule, and iii) heterozygous for one of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule and reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. Detailed Description

[0013] Throughout the specification and claims, various terms related to aspects of the present disclosure are used. Unless otherwise indicated, such terms will be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0014] Unless otherwise explicitly stated, no method or aspect set forth herein is to be construed as requiring that its steps be performed in a particular order. Accordingly, in the claims or specification, when a method claim does not specifically state that the steps are limited to a particular order, no order is to be inferred in any aspect. This applies to any possible basis for non-expression interpretation, including logical matters relative to step arrangement or operational flow, arising from the ordinary meaning derived from grammatical organization or punctuation, or the numbering or type of aspects described in the specification.

[0015] As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents.

[0016] As used herein, the term "about" means that the recited numerical value is an approximation and that small variations will not significantly affect the practice of the disclosed embodiments. In the case of using numerical values, unless the context indicates otherwise, the term "about" means that the numerical value can vary ±10% and still be within the scope of the disclosed embodiments.

[0017] As used herein, in certain embodiments, as needed, the term "comprising" may be replaced by "consisting of" or "consisting essentially of".

[0018] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" or "oligonucleotide" may include polymeric forms of nucleotides of any length, may include DNA and / or RNA, and may be single-stranded, double-stranded or multi-stranded. One strand of a nucleic acid also refers to its complementary sequence.

[0019] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cows, pigs), companion animals (such as, for example, dogs, cats), laboratory animals (such as, for example, mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0020] It has been observed according to the present disclosure that rare ANGPTL3 variant nucleic acid molecules and rare SLC5A2 variant nucleic acid molecules have independent effects on reducing the risk of developing kidney disease. Both rare ANGPTL3 variant nucleic acid molecules and rare SLC5A2 variant nucleic acid molecules result in an increase in estimated glomerular filtration rate (eGFR). Two genes that are expected to increase eGFR may act through the same mechanism / pathway. If this is the case, the effect of a variation in one gene will depend on whether there is also a variation in the second gene. If this is the case, different degrees of effects are expected when the effect of one gene is combined with the effect of the other gene. Surprisingly, the increase in eGFR associated with rare ANGPTL3 variant nucleic acid molecules is independent of the increase in eGFR associated with rare SLC5A2 variant nucleic acid molecules. Thus, subjects who are heterozygous for the ANGPTL3 reference type or an ANGPTL3 variant nucleic acid molecule and heterozygous for the SLC5A2 reference type or an SLC5A2 variant nucleic acid molecule benefit from treatment with inhibitors of two different biological pathways (e.g., with an ANGPTL3 inhibitor and an SLC5A2 inhibitor) to inhibit or prevent kidney disease, alleviate or prevent its symptoms, and / or inhibit or prevent the development of symptoms. It is also believed that such subjects with kidney disease can be further treated with kidney disease therapeutic agents.

[0021] For the purposes of the present disclosure, any particular subject, such as a human, can be classified as having one of the following three ANGPTL3 genotypes: i) ANGPTL3 reference type; ii) heterozygous for an ANGPTL3 variant nucleic acid molecule; or iii) homozygous for an ANGPTL3 variant nucleic acid molecule. A subject is ANGPTL3 reference type when the subject does not have a copy of the ANGPTL3 variant nucleic acid molecule. A subject is heterozygous for an ANGPTL3 variant nucleic acid molecule when the subject has a single copy of the ANGPTL3 variant nucleic acid molecule.

[0022] In any of the embodiments described herein, an ANGPTL3 variant nucleic acid molecule can be any nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) that encodes an ANGPTL3 variant polypeptide having a partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject having an ANGPTL3 polypeptide with a 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 a decrease or abnormality in the 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 codon gain variant, a start codon deletion variant, a stop codon variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 variant polypeptide. In some embodiments, the ANGPTL3 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the ANGPTL3 variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the ANGPTL3 variant nucleic acid molecule contains a variation in the coding region. In some embodiments, the ANGPTL3 variant nucleic acid molecule causes or is predicted to cause premature truncation of the ANGPTL3 polypeptide compared to the reference ANGPTL3. In some embodiments, the ANGPTL3 predicted loss of function or missense variant nucleic acid molecule is a variant predicted to be damaging to protein function by an in vitro prediction algorithm (such as Polyphen, SIFT, or a similar algorithm) (and thus, in this case, protective for humans). In some embodiments, the ANGPTL3 predicted loss of function or missense 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 has an allele frequency of less than 1 per 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 per 1,000 alleles), or any splice site variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant, or in-frame insertion / deletion variant, or other frameshift ANGPTL3 variant.

[0023] For the purposes of the present disclosure, any particular subject, such as a human, can be classified as having one of the following three SLC5A2 genotypes: i) SLC5A2 reference type; ii) heterozygous for an SLC5A2 variant nucleic acid molecule; or iii) homozygous for an SLC5A2 variant nucleic acid molecule. A subject is SLC5A2 reference type when the subject does not have a copy of the SLC5A2 variant nucleic acid molecule. A subject is heterozygous for an SLC5A2 variant nucleic acid molecule when the subject has a single copy of the SLC5A2 variant nucleic acid molecule.

[0024] In some embodiments, the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule.

[0025] In any of the embodiments described herein, an SLC5A2 variant nucleic acid molecule can be any nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) that encodes an SLC5A2 variant polypeptide having a partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject having an SLC5A2 polypeptide with a 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 a decrease or abnormality in the expression or activity of SLC5A2 mRNA or polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is associated with a diminished in vitro response to an SLC5A2 ligand as compared to a reference SLC5A2. In some embodiments, the SLC5A2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 variant 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 contains a single nucleotide polymorphism (SNP). In some embodiments, the SLC5A2 variant nucleic acid molecule contains a variation in the coding region. In some embodiments, the SLC5A2 variant nucleic acid molecule causes or predicts a premature truncation of the SLC5A2 polypeptide as compared to a reference SLC5A2. In some embodiments, an SLC5A2 predicted loss of function or missense variant nucleic acid molecule is a variant predicted to be damaging to protein function by an in vitro prediction algorithm (such as Polyphen, SIFT, or a similar algorithm) (and thus, in such cases, protective for humans). In some embodiments, an SLC5A2 predicted loss of function or missense variant nucleic acid molecule is a variant that causes or predicts a non-synonymous amino acid substitution in the SLC5A2 nucleic acid molecule and has an allele frequency of less than 1 in 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 variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant, or in-frame insertion / deletion variant, or other frameshift SLC5A2 variant.

[0026] For subjects genotyped or determined to be of the ANGPTL3 reference type and the SLC5A2 reference type, such subjects have an increased risk of developing kidney disease. For subjects genotyped or determined to be: i) heterozygous for the ANGPTL3 reference type or an ANGPTL3 variant nucleic acid molecule, and ii) heterozygous for the SLC5A2 reference type or an SLC5A2 variant nucleic acid molecule, such subjects can be treated with an ANGPTL3 inhibitor and an SLC5A2 inhibitor.

[0027] In any of the embodiments described herein, a subject for whom kidney disease is prevented by administration of an ANGPTL3 inhibitor and an SLC5A2 inhibitor can be any subject at risk of developing kidney disease, including but not limited to a subject with a genetic predisposition to developing kidney disease. Other 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 an autoimmune disease. In addition, in some embodiments, any subject can be at risk of developing kidney disease. In some embodiments, administration of an ANGPTL3 inhibitor and an SLC5A2 inhibitor can be carried out to prevent the progression of a subject already suffering from kidney disease to other kidney diseases.

[0028] In any of the embodiments described herein, an ANGPTL3 polypeptide can 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, an SLC5A2 polypeptide can 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.

[0029] In any of the embodiments described herein, using the nucleotide sequence of the ANGPTL3 reference genomic nucleic acid molecule (see ENST00000371129.4 annotated in the Ensembl database (URL: World Wide Web address "useast.ensembl.org / Homo_sapiens / Transcript / Summary?db=core;g=ENSG00000132855;r=1:62597520-62606313;t=ENST00000371129") as a reference sequence, ANGPTL3 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules produced therefrom, or cDNA molecules produced from mRNA molecules) may include variations at positions 62,597,520 - 62,606,313 on chromosome 1. For the ANGPTL3 genomic nucleic acid molecule, the sequence provided by ENST00000371129.4 is only an exemplary sequence. Other sequences of the 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, using the nucleotide sequence of the SLC5A2 reference genomic nucleic acid molecule (see ENST00000330498.4 annotated in the Ensembl database (URL: World Wide Web address "useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000140675;r=16:31483002-31490860;transcript=ENST00000330498.4") as a reference sequence, SLC5A2 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules produced therefrom, or cDNA molecules produced from mRNA molecules) may include variations at positions 31,483,123 - 31,490,769 on chromosome 16. For the SLC5A2 genomic nucleic acid molecule, the sequence provided by ENST00000330498.4 is only an exemplary sequence. Other sequences of 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.

[0031] 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. Combinations of specific variants can form a mask for statistical analysis of the specific associations of ANGPTL3 and SLC5A2 with an increased or decreased risk of developing kidney disease. In some embodiments, the mask for statistical analysis of the specific associations of ANGPTL3 and SLC5A2 with an increased or decreased risk of developing kidney disease can exclude any one or more of these ANGPTL3 variant nucleic acid molecules and / or SLC5A2 variant nucleic acid molecules described herein.

[0032] In any of the embodiments described herein, the subject can 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 renal sclerosis. 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 renal sclerosis.

[0033] Other kidney diseases include, but are not limited to: acquired cystic disease, acute (post-infectious) glomerulonephritis, acute infectious interstitial nephritis, acute interstitial nephritis, acute pyelonephritis, acute renal failure, acute graft failure, acute tubular necrosis, adult polycystic kidney disease, AL amyloidosis, analgesic nephropathy, ANCA-associated vasculitis, anti-glomerular basement membrane disease (Goodpasture 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, BenceNephropathy induced by Jones) protein, benign familial 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-induced nephropathy, crescentic glomerulonephritis, cryoglobulinemia, cystic renal dysplasia, delayed graft function, dense deposit disease, diabetic glomerulosclerosis, diabetic nephropathy, dialysis cystic disease, drug-induced (allergic) acute interstitial nephritis, ectopic kidney, eosinophilic granulomatosis with polyangiitis, Fabry disease, familial juvenile nephronophthisis-medullary cystic disease syndrome, focal segmental glomerulosclerosis (segmental hyalinosis), glomerular cystic disease, glomerulonephritis, glomerulonephritis associated with bacterial endocarditis, glomerulosclerosis, granulomatosis with polyangiitis, hemolytic uremic syndrome, Henoch-Schönlein purpura, hepatitis-associated glomerulonephritis, hereditary nephritis (Alport syndrome), human immunodeficiency virus-associated nephropathy, horseshoe kidney, hydronephrosis, hyperoxaluria, hypertensive nephropathy, immunoglobulin A nephropathy, infantile polycystic kidney disease, ischemic acute tubular necrosis, light chain deposition disease, lupus nephritis, malignant nephrosclerosis, medullary cystic disease, membranoproliferative (mesangiocapillary) glomerulonephritis, membranous glomerulonephritis, membranous nephropathy, mesangial proliferative glomerulonephritis (including Berger disease), microscopic polyangiitis, minimal change glomerular disease, nephritic syndrome, nephroblastoma (Wilms tumor), nephronophthisis (medullary cystic disease syndrome), pigmentary nephropathy, plasma cell dyscrasia (monoclonal immunoglobulin-induced renal injury), polyarteritis nodosa, polycystic kidney disease, proteinuria, pyelonephritis, rapidly progressive (crescentic) glomerulonephritis, renal hypoplasia, renal amyloidosis, renal cell carcinoma, renal dysgenesis, renal dysplasia, renal aplasia, renal infection, renal osteodystrophy, renal calculi (urolithiasis), renal tubular acidosis, renal vasculitis, renovascular hypertension, scleroderma (progressive systemic sclerosis), secondary acquired glomerulonephritis, sepsis-related acute kidney injury, simple renal cyst, 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.

[0034] Symptoms of chronic kidney disease include, but are not limited to, nausea, vomiting, loss of appetite, fatigue and weakness, sleep problems, changes in urine volume, decreased mental sharpness, muscle twitches and cramps, swelling in the feet and ankles, persistent itching, chest pain, fluid accumulation around the lining of the heart, shortness of breath, fluid in the lungs, and uncontrolled high blood pressure (elevated blood pressure).

[0035] Symptoms of kidney stones include, but are not limited to, severe sharp pain in the sides and back below the ribs, pain that radiates to the lower abdomen and groin, wavelike and intense throbbing pain, pain or burning during urination, urine that is pink, red, or brown, cloudy or foul-smelling urine, persistent need to urinate, more frequent urination or less urine output than usual, nausea and vomiting, and fever and chills if there is an infection.

[0036] Symptoms of chronic glomerulonephritis include, but are not limited to, red blood cells in the urine causing the urine to be pink or cola-colored (hematuria), too much protein causing the urine to be foamy (proteinuria), high blood pressure (elevated blood pressure), and fluid retention (edema) with swelling in the face, hands, feet, and abdomen.

[0037] Symptoms of nephronophthisis include, but are not limited to, increased urine volume (polyuria), extreme thirst (polydipsia), general weakness and extreme tiredness (fatigue).

[0038] Symptoms of chronic interstitial nephritis include, but are not limited to, hematuria, fever, increased or decreased urine output, changes in mental state (lethargy, confusion, coma), nausea, vomiting, rash, swelling in any part of the body, and weight gain (due to fluid retention).

[0039] Symptoms of nephrosclerosis include, but are not limited to, impaired vision, hematuria, weight loss, and the accumulation of urea and other nitrogenous wastes in the blood (known as uremia).

[0040] The present disclosure provides methods of treating a subject having or at risk of developing a kidney disease, the methods 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.

[0041] 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 RNAs (siRNAs), and short hairpin RNAs (shRNAs). 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 the ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the ANGPTL3 polypeptide in the cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an antisense molecule that hybridizes to the ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the ANGPTL3 polypeptide in the cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an siRNA that hybridizes to the ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the ANGPTL3 polypeptide in the cells of a subject. In some embodiments, the ANGPTL3 inhibitor comprises an shRNA that hybridizes to the ANGPTL3 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the ANGPTL3 polypeptide in the cells of a subject.

[0042] In some embodiments, the ANGPTL3 antisense nucleic acid molecule comprises or consists of any one of the nucleotide sequences set forth in SEQ ID NOs: 1-325. In some embodiments, the ANGPTL3 siRNA molecule comprises or consists of any one of the nucleotide sequences (sense and antisense strands presented in sequence) set forth in SEQ ID NOs: 326-1189 (e.g., the sense strand is, for example, SEQ ID NO: 326 and the corresponding antisense strand is SEQ ID NO: 327; the sense strand is, for example, SEQ ID NO: 1188 and the corresponding antisense strand is SEQ ID NO: 1189; etc.).

[0043] In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences (sense and antisense strands) described in U.S. Patent 10,995,335 and PCT Publication WO 2019 / 055633, which patents and publications 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) described in U.S. Patent 10,875,884 and PCT Publications WO 2015 / 168589, WO 2015 / 100394, and WO 2011 / 085271, which patents and publications 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) described in U.S. Patents 10,570,393 and 10,337,010 and PCT Publications WO 2016 / 168286 and WO 2012 / 177784, which patents and publications are incorporated herein by reference in their entirety.

[0046] In some embodiments, the ANGPTL3 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at a recognition sequence or a DNA-binding protein that binds to a 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 for 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 comprise or be near 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 nuclease agent targeting a nuclease recognition sequence that comprises or is near the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence that comprises or is near the start codon and one targeting a nuclease recognition sequence that comprises or is near the stop codon, wherein cleavage by the nuclease agents can result in a deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break into 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] Suitable nuclease agents and DNA-binding proteins for use herein include, but are not limited to, zinc finger protein or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary and includes, for example, recognition sequences of about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 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, the CRISPR / Cas system can be used to modify an ANGPTL3 genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex for site-directed cleavage of an ANGPTL3 nucleic acid molecule (comprising a guide RNA (gRNA) complexed with a Cas protein).

[0049] Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with a gRNA. A Cas protein can also comprise a nuclease domain (such as, for example, 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 (such as, for example, FnCpf1). A Cas protein can have full cleavage activity to create a double-strand break in an ANGPTL3 genomic nucleic acid molecule, or it can be a nickase that creates a single-strand break in an ANGPTL3 genomic nucleic acid molecule. Additional 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 (CasB), 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 into a single crRNA. A Cas protein can also be operably linked to a heterologous polypeptide as a fusion protein. For example, a Cas protein can be linked or fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. A Cas protein can be provided in any form. For example, a Cas protein can be provided in the form of a protein (such as a Cas protein complexed with a gRNA). Alternatively, a Cas protein can be provided in the form of a nucleic acid molecule encoding the Cas protein, such as RNA or DNA.

[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 comprise or be near the start codon 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 the stop codon.

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

[0052] A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within the genomic nucleic acid molecule of ANGPTL3. Exemplary gRNAs are gRNAs that effectively direct the Cas enzyme to bind to or cleave the genomic nucleic acid molecule of ANGPTL3, where the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence within the genomic nucleic acid molecule of ANGPTL3. Exemplary gRNAs comprise a DNA targeting segment that hybridizes to a gRNA recognition sequence present within the genomic nucleic acid molecule of ANGPTL3, the gRNA recognition sequence comprising or being near a start codon or a stop codon. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a start codon or located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a stop codon. Suitable gRNAs can comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0053] The Cas protein and the 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 within or outside of a nucleic acid sequence within the target ANGPTL3 genomic nucleic acid molecule that will bind to the DNA targeting segment of the gRNA. For example, formation of a CRISPR complex (comprising a gRNA that hybridizes to a gRNA recognition sequence and is complexed with a Cas protein) can cause one or both strands to be cleaved within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs) a nucleic acid sequence within the ANGPTL3 genomic nucleic acid molecule that will bind to the DNA targeting segment of the gRNA.

[0054] Such methods can generate ANGPTL3 genomic nucleic acid molecules in which, for example, regions of the ANGPTL3 genomic nucleic acid molecule are disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell can also be contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the ANGPTL3 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein can generate 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 comprising amino acids 32 to 41 of ANGPTL3 (i.e., EPKSRFAMLD; SEQ ID NO: 3738) (see 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 WO 2020 / 243031, which is incorporated herein by reference in its entirety.

[0057] In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or an antigen-binding fragment thereof that specifically binds and neutralizes, inhibits, blocks, eliminates, reduces, or interferes with at least one activity of ANGPTL3, particularly human ANGPTL3 (SEQ ID NO: 3739). The activities of ANGPTL3 that can be neutralized, inhibited, blocked, eliminated, reduced, or interfered with by the antibodies or fragments of the present disclosure include, but are not limited to, inhibiting LPL activity, inducing angiogenesis, etc. In some embodiments, the antibody or fragment thereof can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of ANGPTL3 by binding to an epitope of ANGPTL3 that is directly involved in the targeting activity of ANGPTL3. In some embodiments, the antibody or fragment thereof can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of ANGPTL3 by binding to an epitope of ANGPTL3 that is not directly involved in the targeting activity of ANGPTL3, but the antibody or fragment that binds to this epitope inhibits, blocks, eliminates, reduces, or interferes with the targeting activity of ANGPTL3 spatially or conformationally. In some embodiments, the antibody or fragment thereof binds to an epitope of ANGPTL3 that is not directly involved in the targeting activity of ANGPTL3 (e.g., inhibiting LPL activity, inducing angiogenesis, etc.) (i.e., a non-blocking antibody), but the antibody or fragment that binds to this epitope results in enhanced clearance of ANGPTL3 from the circulation compared to the clearance of ANGPTL3 in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, eliminating, reducing, or interfering with the activity of ANGPTL3. The clearance of ANGPTL3 from the circulation can be particularly enhanced by a combination of two or more different non-blocking antibodies that do not compete with each other for specific binding to ANGPTL3.

[0058] The antibody (Ab) can be full-length (e.g., an IgG1 or IgG4 antibody) or can contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and can be modified to affect function, e.g., to eliminate residual effector functions (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

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

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

[0061] In some embodiments, the antibody or its fragment comprises a pair of HCVR and LCVR sequences (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 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 its fragment comprises a pair of HCVR and LCVR sequences selected from the group consisting of SEQ ID NO: 3740 / 3751, 3741 / 3752, 3742 / 3753, 3744 / 3755, 3745 / 3756, 3747 / 3758, and 3750 / 3761. In some embodiments, the antibody or its fragment comprises the HCVR and LCVR sequence pair of SEQ ID NO: 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 and a light chain CDR3 (LCDR3) amino acid sequence, the HCDR3 amino acid sequence being selected from the group consisting of SEQ ID NO: 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, and 3772, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; the LCDR3 amino acid sequence being selected from the group consisting of SEQ ID NO: 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, and 3783, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the antibody or fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair comprising SEQ ID NO: 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 NO: 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 NO: 3766 / 3777.

[0063] In some embodiments, the antibody or fragment thereof further comprises a heavy chain CDR1 (HCDR1) amino acid sequence and a heavy chain CDR2 (HCDR2) amino acid sequence, wherein the HCDR1 amino acid sequence is selected from the group consisting of SEQ ID NO: 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, and 3794, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; the HCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO: 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, and 3805, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and optionally further comprises a light chain CDR1 (LCDR1) amino acid sequence and / or a light chain CDR2 (LCDR2) amino acid sequence, wherein the LCDR1 amino acid sequence is selected from the group consisting of SEQ ID NO: 3806, 3807, 3808, 3809, 3810, 3811, 3812, 3813, 3814, 3815, and 3816, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; the LCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO: 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 / HCDR2 / HCDR3 combination and / or an LCDR1 / LCDR2 / LCDR3 combination, the HCDR1 / HCDR2 / HCDR3 combination selected from the group consisting of: SEQ ID NO: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; the LCDR1 / LCDR2 / LCDR3 combination selected from the group consisting of: SEQ ID NO: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 chain CDR amino acid sequences and the light chain CDR amino acid sequences comprise a CDR sequence combination selected from the group consisting of: SEQ ID NO: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 / 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 heavy chain CDR amino acid sequences and the light chain CDR amino acid sequences comprise CDR sequence combinations of SEQ ID NO: 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 chain CDR amino acid sequences and the light chain CDR amino acid sequences comprise the CDR sequence combination of SEQ ID NO: 3788 / 3799 / 3766 / 3810 / 3821 / 3777.

[0065] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds ANGPTL3 comprises heavy chain CDR domains and light chain CDR domains, and the heavy chain CDR domains and light chain CDR domains are contained within an HCVR / LCVR pair selected from the group consisting of: SEQ ID NO: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 the specific HCVR and / or LCVR amino acid sequences disclosed herein. Conventional definitions that can be applied to identify the boundaries of CDRs include the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the positions of structural loop regions, and the AbM definition is a compromise method between the Kabat and Chothia methods. 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 can also be used to identify CDR sequences within antibodies. In some embodiments, the antibody or fragment thereof comprises a CDR sequence that is contained within an HCVR and LCVR pair of SEQ ID NO:3740 / 3751, 3741 / 3752, 3742 / 3753, 3744 / 3755, 3745 / 3756, 3747 / 3758, or 3750 / 3761. In some embodiments, the antibody or fragment thereof comprises a CDR sequence that is contained within an HCVR and LCVR pair of SEQ ID NO:3744 / 3755.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment comprising a heavy-chain CDR sequence and a light-chain CDR sequence for specific binding to ANGPTL3, wherein the heavy-chain CDR sequence and the light-chain CDR sequence are included in the HCVR / LCVR sequence pairs of SEQ ID NO: 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 with an antibody or fragment thereof comprising the HCVR / LCVR sequence pair of SEQ ID NO: 3744 / 3755 for specific binding to ANGPTL3. In some embodiments, the antibody or antigen-binding fragment thereof competes with an antibody or fragment thereof comprising a combination of heavy-chain CDR sequences and light-chain CDR sequences 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 / 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 with an antibody or fragment thereof comprising a combination of heavy-chain CDR sequences and light-chain CDR sequences of SEQ ID NO: 3788 / 3799 / 3766 / 3810 / 3821 / 3777 for specific binding to ANGPTL3.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on ANGPTL3 as identified by such an antibody or fragment thereof, where the antibody or fragment thereof comprises heavy chain CDR sequences and light chain CDR sequences from the HCVR / LCVR sequence pairs of SEQ ID NO: 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 as identified by such an antibody or fragment thereof, where the antibody or fragment thereof comprises the HCVR / LCVR sequence pair of SEQ ID NO: 3744 / 3755. In some embodiments, the antibody or fragment thereof binds to the same epitope on ANGPTL3 as identified by such an antibody or fragment thereof, where the antibody or fragment thereof comprises a combination of heavy chain CDR sequences and light chain CDR sequences 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 / 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, such an epitope is identified by an antibody or fragment thereof comprising the combination of heavy chain CDR sequences and light chain CDR sequences of SEQ ID NO: 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 to 209 of SEQ ID NO: 3739 and neutralizes, inhibits, eliminates, 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, eliminates, 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 to 200, 17 to 100, 17 to 70, 17 to 65, 17 to 60, 17 to 57, or 17 to 50 of ANGPTL3 (SEQ ID NO: 3739), optionally provided that 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 to 200, 40 to 100, 40 to 70, 50 to 200, 50 to 100, 50 to 70, 58 to 200, 58 to 100, 58 to 70, 58 to 68, or 61 to 66 of ANGPTL3 (SEQ ID NO: 3739), optionally provided that the antibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQ ID NO: 3828. In some embodiments, the epitope to which the antibody or antibody fragment binds can involve more than one of the recited epitopes or residues within the N-terminal coiled-coil region of ANGPTL3, optionally provided that 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 NO: 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 an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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 an 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 an LCVR encoded by the nucleic acid sequence of SEQ ID NO: 3844.

[0071] In some embodiments, the antibody or fragment thereof comprises an HCVR / LCVR (HCVR / LCVR) sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NO: 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 NO: 3829 / 3840, 3830 / 3841, 3831 / 3842, 3833 / 3844, 3834 / 3845, 3836 / 3847, or 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 NO: 3833 / 3844.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 domain and an LCDR3 domain, wherein the HCDR3 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858, 3859, 3860, and 3861, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto; the LCDR3 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3862, 3863, 3864, 3865, 3866, 3867, 3868, 3869, 3870, 3871, and 3872, 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 HCDR3 and LCDR3 sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NO: 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 NO: 3851 / 3862, 3852 / 3863, 3853 / 3864, 3855 / 3866, 3856 / 3867, 3858 / 3869, or 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 NO: 3855 / 3866.

[0073] In some embodiments, the antibody or fragment thereof further comprises an HCDR1 domain and an HCDR2 domain, wherein the HCDR1 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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; the HCDR2 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3884, 3885, 3886, 3887, 3888, 3889, 3890, 3891, 3892, 3893, and 3894, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto; and optionally further comprises an LCDR1 domain and / or an LCDR2 domain, wherein the LCDR1 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3895, 3896, 3897, 3898, 3899, 3900, 3901, 3902, 3903, 3904, and 3905, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto; the LCDR2 domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 comprises an HCDR1 / HCDR2 / HCDR3 combination and / or an LCDR1 / LCDR2 / LCDR3 combination, wherein the HCDR1 / HCDR2 / HCDR3 combination is encoded by a nucleotide sequence combination selected from the group consisting of: SEQ ID NO: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 the LCDR1 / LCDR2 / LCDR3 combination is encoded by a nucleotide sequence combination selected from the group consisting of: SEQ ID NO: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 chain CDR sequences and light chain CDR sequences encoded by the nucleotide sequence combination of SEQ ID NO:3877 / 3888 / 3855 / 3899 / 3910 / 3866.

[0075] In some embodiments, the anti-ANGPTL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR is encoded by nucleotide sequence segments derived from V H , D H and J H germline sequences, and the LCVR is encoded by nucleotide sequence segments derived from V K and J K germline sequences, wherein the HCVR and LCVR are encoded by nucleotide sequence segments derived from a germline gene combination 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) V H 3-11, D H 1-1, J H 4, V K 1-39 and J K4; (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 equilibrium dissociation constant (K D ) for specific binding of the antibody or its antigen - binding fragment to ANGPTL3 is 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 surface plasmon resonance (e.g., BIACORE TM ). In some embodiments, the antibody exhibits a K D 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.

[0077] In some embodiments, the anti - ANGPTL3 antibody has a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites, or for example, removal of fucose moieties to increase antibody - dependent cell - mediated 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 unwanted immune responses against the therapeutic antibody, or increase the antibody's affinity. In still other applications, galactosylation can be modified to alter 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 the SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the SLC5A2 polypeptide in the cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an antisense molecule that hybridizes to the SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the SLC5A2 polypeptide in the cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an siRNA that hybridizes to the SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the SLC5A2 polypeptide in the cells of a subject. In some embodiments, the SLC5A2 inhibitor comprises an shRNA that hybridizes to the SLC5A2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the SLC5A2 polypeptide in the cells of a subject.

[0080] In some embodiments, the SLC5A2 antisense nucleic acid molecule comprises or consists of any one of the nucleotide sequences represented by SEQ ID NOs: 1190-1569. In some embodiments, the SLC5A2 siRNA molecule comprises or consists of any one of the nucleotide sequences represented by SEQ ID NOs: 1570-3737 (the sense strand and the antisense strand presented in sequence) (e.g., the sense strand is, for example, SEQ ID NO: 1570 and the corresponding antisense strand is SEQ ID NO: 1571; the sense strand is, for example, 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 incisions or double-strand breaks at an recognition sequence or a DNA-binding protein that binds to an recognition sequence within the SLC5A2 genomic nucleic acid molecule. The recognition sequence can be located within the coding region of the SLC5A2 gene or in a regulatory region that affects gene expression. The recognition sequence for the DNA-binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein-coding region. The recognition sequence can comprise or be near the start codon of the SLC5A2 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 nuclease agent targeting a nuclease recognition sequence that comprises or is near the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence that comprises or is near the start codon and one targeting a nuclease recognition sequence that comprises or is near the stop codon, wherein cleavage by the nuclease agents can result in a deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces an incision or double-strand break at a desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

[0082] Suitable nuclease agents and DNA-binding proteins 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 interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary and includes, for example, recognition sequences of about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 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 within a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex for site-directed cleavage of the SLC5A2 nucleic acid molecule (comprising a guide RNA (gRNA) complexed with a Cas protein).

[0084] Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with a gRNA. The Cas protein may also comprise a nuclease domain (such as, for example, a DNA nuclease or an RNA nuclease 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 (such as, for example, FnCpf1). The Cas protein can have full cleavage activity to generate a double-strand break in the SLC5A2 genomic nucleic acid molecule, or it can be a nickase that generates a single-strand break in the SLC5A2 genomic nucleic acid molecule. Additional 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 (CasB), 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 into a single crRNA. The Cas protein can also be operably linked to a heterologous polypeptide as a fusion protein. For example, the Cas protein can be linked or fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. The Cas protein can be provided in any form. For example, the Cas protein can be provided in the form of a protein (such as a Cas protein complexed with a gRNA). Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule encoding the Cas protein, such as RNA or DNA.

[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 in the SLC5A2 genomic nucleic acid molecule. The gRNA recognition sequence can comprise or be near 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 codon or stop codon.

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

[0087] A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within the genomic nucleic acid molecule of SLC5A2. An exemplary gRNA is a gRNA that effectively directs the Cas enzyme to bind to or cleave the genomic nucleic acid molecule of SLC5A2, wherein the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence within the genomic nucleic acid molecule of SLC5A2. An exemplary gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence present within the genomic nucleic acid molecule of SLC5A2, the gRNA recognition sequence comprising or being near a start codon or a stop codon. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a start codon or located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a stop codon. Suitable gRNAs can comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0088] The Cas protein and the 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 within or outside of a nucleic acid sequence in the target SLC5A2 genomic nucleic acid molecule that will bind to the DNA targeting segment of the gRNA. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes to a gRNA recognition sequence and is complexed with a Cas protein) can cause one or both strands to be cleaved within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs) a nucleic acid sequence in the SLC5A2 genomic nucleic acid molecule that will bind to the DNA targeting segment of the gRNA.

[0089] Such methods can generate SLC5A2 genomic nucleic acid molecules in which, for example, regions of the SLC5A2 genomic nucleic acid molecule are disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell can also be contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the SLC5A2 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein can generate two or more double-strand breaks or two or more single-strand breaks.

[0090] In some embodiments, the SLC5A2 inhibitor targets the SGLT2 polypeptide (the polypeptide encoded by the SLC5A2 gene). In some embodiments, the SLC5A2 inhibitor includes INVO (canagliflozin), (dapagliflozin), (empagliflozin), (ipragliflozin), (luseogliflozin) or APLEWA (tofogliflozin) or any combination thereof. In some embodiments, the SLC5A2 inhibitor includes canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin or tofogliflozin or any combination thereof. In some embodiments, the SLC5A2 inhibitor includes canagliflozin. In some embodiments, the SLC5A2 inhibitor includes dapagliflozin. In some embodiments, the SLC5A2 inhibitor includes empagliflozin. In some embodiments, the SLC5A2 inhibitor includes ipragliflozin. In some embodiments, the SLC5A2 inhibitor includes luseogliflozin. In some embodiments, the SLC5A2 inhibitor includes tofogliflozin.

[0091] Inhibitory nucleic acid molecules can include RNA, DNA, or both RNA and DNA. Inhibitory nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence (such as a heterologous nucleic acid sequence in a vector) or a heterologous label. For example, an inhibitory nucleic acid molecule can be within a vector containing the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence or as an exogenous donor sequence containing the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. Inhibitory nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where an enzyme-dependent secondary signal generation occurs. The term "label" can also refer to a "tag" or a hapten that can selectively bind to a conjugate molecule such that the conjugate molecule is used to generate a detectable signal when added together with a substrate subsequently. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag, and a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorescent substrate is used for examination 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, an epitope tag, or the Fc portion of an immunoglobulin. Many labels include, for example, particles, fluorophores, haptens, enzymes, and their calorimetric, fluorescent, and chemiluminescent substrates and other labels.

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

[0093] The inhibitory nucleic acid molecule may also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to 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, but are not limited to, 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-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy, and other 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-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0094] Nucleotide analogs may also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of 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 the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C 1-10 alkyl or C 2-10 alkenyl and C 2-10 alkynyl. Exemplary 2'-sugar modifications also include, but are not limited to, -O[(CH2) 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 1-10alkyl, 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 cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, specifically at the 3'-position of the sugar on the 3'-terminal nucleotide or at the 3'-position of the sugar and the 5'-position of the 5'-terminal nucleotide in a 2'-5'-linked oligonucleotide. Modified sugars can also include those having modifications (such as CH2 and S) at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimetics that replace furanose, such as a cyclobutyl moiety.

[0095] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, phosphate moieties that can be modified such that the linkage between two nucleotides contains the following: phosphorothioate, chiral phosphorothioate, dithiophosphate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphonite, aminophosphate (including 3'-aminoaminophosphate and aminoalkylaminophosphate), thiocarbonylaminophosphate, thiocarbonylalkyl phosphonate, thiocarbonylalkyl phosphotriester, and borane phosphate. These phosphate or modified phosphate linkages between two nucleotides can be 3'-5' linkages or 2'-5' linkages, and the linkages can have an inverted polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Nucleotide replacements also include peptide nucleic acid (PNA).

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

[0097] In some embodiments, the siRNA molecule has terminal modifications. 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)-vinylphosphonate is used.

[0098] In some embodiments, the siRNA molecule has backbone modifications. In some embodiments, modified phosphodiester groups that link consecutive ribonucleosides have been shown to enhance the stability and in vivo bioavailability of siRNA. The non-ester groups (-OH, =O) of the phosphodiester linkage can be replaced with sulfur, boron, or acetate to give phosphorothioate, boranophosphate, and phosphonoacetate linkages. Additionally, replacing the phosphodiester group with a phosphotriester can facilitate cellular uptake of siRNA and retention on serum components by eliminating their negative charge. In some embodiments, the siRNA molecule has sugar modifications. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exo- and endonucleases), whereby the 2'-hydroxy group can act as a nucleophile and attack the adjacent phosphorus in the phosphodiester bond. Such alternatives 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 base can be replaced with a modified base such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0100] In some embodiments, the siRNA molecule is conjugated to a lipid. The lipid can be conjugated to the 5' or 3' end of the siRNA to improve its in vivo bioavailability by allowing them to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, as well as fatty acids such as palmitate and tocopherol.

[0101] In some embodiments, a representative siRNA has the following formula:

[0102] Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN /

[0103] Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N

[0104] wherein: "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification, "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0105] In any of the embodiments described herein for antisense molecules and siRNA molecules, the molecule can comprise 1, 2, or 3 additional nucleotides at the 5'-end, 3'-end, or both the 5'-end and 3'-end. In some embodiments, the antisense molecule and the siRNA molecule comprise 1, 2, or 3 additional nucleotides at the 5'-end. In some embodiments, the antisense molecule and the siRNA molecule comprise 1, 2, or 3 additional nucleotides at the 3'-end. In some embodiments, the antisense molecule and the siRNA molecule comprise 1, 2, or 3 additional nucleotides at both the 5'-end and 3'-end.

[0106] In any of the embodiments described herein for antisense molecules and siRNA molecules, the molecule can comprise a substantially identical sequence 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 molecule and the siRNA molecule have at least 80% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense molecule and the siRNA molecule have at least 85% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense molecule and the siRNA molecule have at least 90% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense molecule and the siRNA molecule have at least 95% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense molecule and the siRNA molecule have at least 98% homology to the nucleotide sequences disclosed herein. In some embodiments, the antisense molecule and the siRNA molecule have at least 99% homology to the nucleotide sequences disclosed herein.

[0107] In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered, for example, as a one- to two-hour intravenous infusion or subcutaneous injection. In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered at a dose level ranging from about 50 mg to about 900 mg, about 100 mg to about 800 mg, about 150 mg to about 700 mg, or about 175 mg to about 640 mg (2.5 mg / kg to 9.14 mg / kg; 92.5 to 338 mg / m 2 – based on the assumption of a body weight of 70 kg and the conversion of the mg / kg dose level to the mg / m dose level according to the mg / kg dose multiplier value for a human of 37 2 dose level conversion).

[0108] The present disclosure also provides a vector 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 nucleic acid molecules. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector into 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 (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YAC), Epstein-Barr virus (EBV)-derived episomes, and other expression vectors known in the art.

[0109] The present disclosure also provides a composition 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 an 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 helices, and lipid microtubes. The carrier can include a buffered salt solution, such as PBS, HBSS, etc.

[0110] In some embodiments, these methods further comprise detecting the presence or absence of an ANGPTL3 variant nucleic acid molecule in a biological sample from a subject, and detecting the presence or absence of an SLC5A2 variant nucleic acid molecule. 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, these methods further comprise administering a kidney disease therapeutic agent to a subject who is: i) homozygous for the ANGPTL3 reference type or an ANGPTL3 variant nucleic acid molecule, and ii) homozygous for the SLC5A2 reference type or an SLC5A2 variant nucleic acid molecule.

[0111] The present disclosure also provides a composition comprising a combination of any one of an ANGPTL3 inhibitor and an SLC5A2 inhibitor with a second therapeutic agent. The second therapeutic agent can be one or more of any agents such as the following: (1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and the like; (2) inhibitors of cholesterol uptake and / or bile acid reabsorption; (3) niacin, which increases lipoprotein catabolism; (4) fibrates or amphipathic carboxylic acids, which reduce low-density lipoprotein (LDL) levels, increase high-density lipoprotein (HDL) and TG levels, and reduce the number of non-fatal heart attacks; and (5) activators of LXR transcription factors, which play a role in cholesterol (such as 22-hydroxy cholesterol) elimination, or fixed combinations such as ezetimibe plus simvastatin; statins with bile resins (e.g., cholestyramine, colestipol, colesevelam), fixed combinations of niacin plus statins (e.g., niacin with lovastatin); or fixed combinations with other lipid-lowering agents such as ethyl omega-3 fatty acids (e.g., omacor). In addition, the second therapeutic agent can be one or more other inhibitors of ANGPTL3 and inhibitors of other molecules (such as ANGPTL4, ANGPTL5, ANGPTL6, and proprotein convertase subtilisin / kexin type 9 (PCSK9)), which are involved in lipid metabolism, particularly cholesterol and / or triglyceride homeostasis. Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity.

[0112] The present disclosure also provides methods of treating a subject with a therapeutic agent for kidney disease. In some embodiments, the subject has a kidney disease. In some embodiments, the subject is at risk of developing a kidney disease. These methods include administering a therapeutic agent for kidney disease to the subject. These methods include determining whether the subject has an ANGPTL3 variant nucleic acid molecule and whether the subject has an SLC5A2 variant nucleic acid molecule. This determination can be made by obtaining or having obtained a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine the genotype of the subject that includes the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. These methods include administering or continuing to administer a therapeutic agent for kidney disease to a subject who is homozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. These methods include administering or continuing to administer a therapeutic agent for kidney disease and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who is: i) heterozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, ii) heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and of a reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or iii) of an ANGPTL3 reference type and an SLC5A2 reference type. The presence of the genotype of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule indicates a reduced risk of the subject developing a kidney disease.

[0113] 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.

[0114] For a subject who is genotyped or determined to be: i) of an ANGPTL3 reference type or heterozygous for an ANGPTL3 variant nucleic acid molecule, and ii) of an SLC5A2 reference type or heterozygous for an SLC5A2 variant nucleic acid molecule, an ANGPTL3 inhibitor and an SLC5A2 inhibitor as described herein can be administered to such a subject.

[0115] In some embodiments, the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion 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 codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

[0116] Detecting the presence or absence of an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule in a biological sample from a 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 molecule can be present within cells obtained from the subject.

[0117] In some embodiments, the subject is heterozygous for an ANGPTL3 reference or an ANGPTL3 variant nucleic acid molecule and heterozygous for an SLC5A2 reference or an SLC5A2 variant nucleic acid molecule, and a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor is administered or continued to be administered to the subject. The ANGPTL3 inhibitor and the SLC5A2 inhibitor can be any inhibitor described herein or any combination thereof.

[0118] In some embodiments, the method of treatment or prevention includes detecting the presence or absence of an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide in a biological sample from a subject. In some embodiments, when the subject does not have an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide, a kidney disease therapeutic agent is administered to the subject and an ANGPTL3 inhibitor and an SLC5A2 inhibitor are administered.

[0119] The present disclosure also provides methods of treating a subject with a therapeutic agent for kidney disease, wherein the subject has a kidney disease or is at risk of developing a kidney disease. In some embodiments, the methods include determining whether the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide by obtaining or having obtained a biological sample from the subject and 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. When the subject does not have an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide, administering or continuing to administer the therapeutic agent for kidney disease and / or administering to the subject an ANGPTL3 inhibitor and an SLC5A2 inhibitor. The presence of the ANGPTL3 variant polypeptide and the SLC5A2 variant polypeptide indicates a reduced risk of the subject developing a kidney disease. In some embodiments, the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide. In some embodiments, the subject does not have an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide.

[0120] Detecting the presence or absence of an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide in a biological sample from a subject and / or determining whether the subject has an ANGPTL3 variant polypeptide and an SLC5A2 variant polypeptide can be performed by any of the methods described herein. In some embodiments, the methods can be performed in vitro. In some embodiments, the methods can be performed in situ. In some embodiments, the methods can be performed in vivo. In any of these embodiments, the polypeptide can be present within cells obtained from the subject.

[0121] Examples of therapeutic agents for kidney disease 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), antihypertensive agents, phosphate binders, sodium bicarbonate, cholesterol drugs, and gliflozin, or any combination thereof.

[0122] Examples of therapeutic agents for treating or inhibiting kidney diseases associated with 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.

[0123] Examples of therapeutic agents for treating or inhibiting 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), β-adrenergic blockers (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), α-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), corticosteroids (e.g., prednisone), and immunosuppressants (e.g., cyclophosphamide), or any combination thereof. (diltiazem)), β-adrenergic blockers (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), α-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), corticosteroids (e.g., prednisone), and immunosuppressants (e.g., cyclophosphamide), or any combination thereof.

[0124] Examples of therapeutic agents for treating or inhibiting nephronophthisis include, but are not limited to, erythropoietin and antihypertensive drugs, or any combination thereof.

[0125] Examples of therapeutic agents for treating or suppressing kidney diseases of chronic interstitial nephritis include, but are not limited to, corticosteroids, erythropoietin, antihypertensive drugs, statins, and chelating agents (e.g., succimer and calcium disodium edetate), or combinations thereof.

[0126] Examples of therapeutic agents for treating or suppressing kidney diseases of renal sclerosis 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), β-adrenergic blockers (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), α-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), renin inhibitors (e.g., aliskiren), vasodilators (e.g., minoxidil and hydralazine), and α-1 blockers (e.g., doxazosin), or any combination thereof. (diltiazem)), β-adrenergic blockers (e.g., metoprolol, bisoprolol, esmolol, atenolol, propranolol, sotalol, labetalol, pindolol, and penbutolol), α-adrenergic agonists (e.g., clonidine, tizanidine, and dexmedetomidine), renin inhibitors (e.g., aliskiren), vasodilators (e.g., minoxidil and hydralazine), and α-1 blockers (e.g., doxazosin), or any combination thereof.

[0127] In some embodiments, 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, the dose of a kidney 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% 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 the kidney disease therapeutic agent can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Additionally, compared to a subject who is homozygous for an ANGPTL3 variant nucleic acid molecule and homozygous for an ANGPTL3 variant nucleic acid molecule, 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 can be administered at a lower frequency.

[0128] The administration of the kidney disease therapeutic agent, ANGPTL3 inhibitor, and / or SLC5A2 inhibitor can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated one, two, three, four, five, six, seven, eight, nine, ten, or more times. For example, according to certain dosing regimens, a subject can receive treatment for a longer period, such as 6 months, 1 year, or longer.

[0129] The administration of the kidney disease therapeutic agent, ANGPTL3 inhibitor, and / or SLC5A2 inhibitor can be carried out by any suitable route, including but not limited to parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is desirably sterile and substantially isotonic and is manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dose for a single administration). The pharmaceutical composition 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 substantially harmless to its recipient.

[0130] As used herein, the terms "treat", "treating", "treatment" and "prevent", "preventing", "prevention" respectively refer to eliciting a desired biological response, such as a therapeutic effect and a preventive effect. In some embodiments, the therapeutic effect includes one or more of the following: after administration of an agent or a composition comprising the agent, the kidney disease is attenuated / mitigated, the severity of the kidney disease is attenuated / mitigated (such as, reduction or inhibition of the development of the kidney disease), the symptoms and the effects associated with the kidney disease are attenuated / mitigated, the onset of the symptoms and the effects associated with the kidney disease is delayed, the severity of the symptoms associated with the kidney disease is reduced, the number of the symptoms and the effects associated with the kidney disease is reduced, the latency of the symptoms and the effects associated with the kidney disease is shortened, the symptoms and the effects associated with the kidney disease are improved, the secondary symptoms are reduced, the secondary infections are reduced, the recurrence of the kidney disease is prevented, the number or frequency of the recurrence attacks is reduced, the latency between the symptom attacks is prolonged, the time to continuous progression is increased, the remission is accelerated, or the efficacy of an alternative therapy is increased or the resistance to the alternative therapy is reduced and / or the survival time of the affected host animal is prolonged. The preventive effect may include, after administration of a treatment regimen, completely or partially avoiding / inhibiting or delaying the development / progression of the kidney disease (such as, completely or partially avoiding / inhibiting or delaying), and increasing the survival time of the affected host animal. The treatment of the kidney disease encompasses the treatment of a subject who has been diagnosed with any form of kidney disease at any clinical stage or manifestation, the delay of the onset or evolution or exacerbation or deterioration of the symptoms or signs of the kidney disease, and / or the prevention and / or mitigation of the severity of the kidney disease.

[0131] The present disclosure also provides methods for identifying a subject at increased risk of developing a kidney disease. These methods include 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 in the biological sample. When the subject is ANGPTL3 reference type and SLC5A2 reference type, then the subject is at increased risk of developing a kidney disease. When 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 when the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, then the subject is at reduced risk of developing a kidney disease.

[0132] 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.

[0133] In some embodiments, the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide. In some embodiments, the ANGPTL3 variant nucleic acid molecule is any one of the ANGPTL3 variant nucleic acid molecules described herein.

[0134] In some embodiments, the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide. In some embodiments, the SLC5A2 variant nucleic acid molecule is any one of the SLC5A2 variant nucleic acid molecules described herein.

[0135] Compared with a subject having no copies of the ANGPTL3 variant nucleic acid molecule and no copies of the SLC5A2 variant nucleic acid molecule, a subject having a single copy of the ANGPTL3 variant nucleic acid molecule and a single copy of the SLC5A2 variant nucleic acid molecule is more protected from developing kidney disease. Without being bound by any particular theory or mechanism of action, it is believed that a single copy of the ANGPTL3 variant nucleic acid molecule and a single copy of the SLC5A2 variant nucleic acid molecule (i.e., a heterozygote of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or a heterozygote of one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and a reference form of the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule) protects the subject from developing kidney disease, and it is also believed that having two copies of the ANGPTL3 variant nucleic acid molecule and two copies of the SLC5A2 variant nucleic acid molecule (i.e., a homozygote of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule) can better protect the subject from developing kidney disease relative to a subject having a single copy of the foregoing variant nucleic acid molecule. Thus, in some embodiments, a single copy of the ANGPTL3 variant nucleic acid molecule and a single copy of the SLC5A2 variant nucleic acid molecule may not be fully protective, but may protect the subject from developing kidney disease partially or incompletely. While not wishing to be bound by any particular theory, there may be other factors or molecules involved in the development of kidney disease that are still present in a subject having a single copy of the ANGPTL3 variant nucleic acid molecule and a single copy of the SLC5A2 variant nucleic acid molecule, resulting in a failure to provide complete protection from developing kidney disease.

[0136] 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 molecule can be present within cells obtained from the subject.

[0137] In some embodiments, these methods further comprise administering to a subject a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor, wherein the subject 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 an ANGPTL3 nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises siRNA. 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. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises siRNA. In some embodiments, the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin or any combination thereof.

[0138] The present disclosure also provides a method for determining the total burden or polygenic risk score (PRS) of a subject having 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 can be used in an association analysis 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 of the reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. When the subject has a lower total burden, the risk of the subject developing kidney disease increases, and a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor are administered to or continued to be administered to the subject. When the subject has a higher total burden, the risk of the subject developing kidney disease decreases, and a kidney disease therapeutic agent is administered to or continued to be administered to the subject. The higher the total burden, the lower the risk of developing kidney disease.

[0139] In some embodiments, the total burden of any two or more ANGPTL3 and SLC5A2 variant nucleic acid molecules in the subject represents the weighted sum of multiple any 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 or greater than 1,000,000 genetic variants present in or around (up to 10 Mb) the ANGPTL3 gene and the SLC5A2 gene, wherein the genetic burden is the number of alleles multiplied by the association estimate with kidney disease or the relevant outcome for each allele (e.g., weighted polygenic burden score). In some embodiments, when the total burden of the subject is higher than a desired threshold score, the risk of the subject developing kidney disease decreases. In some embodiments, when the total burden of the subject is lower than a desired threshold score, the risk of the subject developing kidney disease increases.

[0140] In some embodiments, the total load can be divided into quintiles, such as the highest quintile, the second quintile, the middle quintile, the fourth quintile, and the lowest quintile, where the highest quintile of the total load corresponds to the lowest risk group, and the lowest quintile of the total load corresponds to the highest risk group. In some embodiments, subjects with a higher total load include the highest weighted total load, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of the total load of the subject population. In some embodiments, genetic variants include genetic variants associated with kidney disease in the top 10%, top 20%, top 30%, top 40%, or top 50% of the associated p-value range. In some embodiments, each of the identified genetic variants includes a genetic variant associated with kidney disease, and its p-value is not greater than about 10 -2 、about 10 -3 、about 10 -4 、about 10 -5 、about 10 -6 、about 10 -7 、about 10 -8 、about 10 -9 、about 10 -10 、about 10 -11 、about 10 -12 、about 10 -13 、about 10 -14 or about 10 -15 。In some embodiments, the identified genetic variants include genetic variants associated with kidney disease, and their p-values are less than 5x10 -8 。In some embodiments, the identified genetic variants include genetic variants associated with kidney disease in high-risk subjects, with the following odds ratios (OR) compared to the rest of the reference population, for the top 20% of the distribution being about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater; 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. In some embodiments, the range of the odds ratio (OR) can be from about 1.0 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, from about 4.5 to about 5.0, from about 5.0 to about 5.5, from about 5.5 to about 6.0, from about 6.0 to about 6.5, from about 6.5 to about 7.0, or greater than 7.0. In some embodiments, high-risk subjects have the lowest decile, quintile, or tertile of the total load in the reference population. The threshold of the total load can be determined according to the nature of the expected actual application and the risk difference that would be considered meaningful for the actual application.

[0141] The present disclosure also provides methods for detecting the presence or absence of ANGPTL3 and SLC5A2 variant nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules produced from mRNA molecules) in a biological sample from a subject. It should be understood that gene sequences within a population and the mRNA molecules encoded by such genes can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0142] The 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, a tumor biopsy, a fine needle aspirate, or a body fluid sample, such as blood, gingival crevicular fluid, plasma, serum, lymph fluid, ascites, cyst fluid, or urine. In some cases, the sample includes an oral 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 employed. For example, when detecting any ANGPTL3 and SLC5A2 variant nucleic acid molecules, a preliminary treatment can be employed that is designed to isolate or enrich genomic DNA from the biological sample. A variety of techniques can be used for this purpose. When detecting the levels of any ANGPTL3 and SLC5A2 variant nucleic acid molecules, different techniques can be used to enrich the biological sample for mRNA molecules. A variety of methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0143] In some embodiments, detecting ANGPTL3 and SLC5A2 variant nucleic acid molecules in a subject includes 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 produced from the mRNA molecules in the biological sample are present in the sample. In some embodiments, these methods detect ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules containing any of the genetic variants described herein, or mRNA molecules produced therefrom, or cDNA molecules produced from the mRNA molecules.

[0144] In some embodiments, methods for detecting the presence or absence of ANGPTL3 and SLC5A2 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules produced from mRNA molecules) in a subject include performing an assay on a biological sample obtained from the subject. The assay determines whether the nucleic acid molecules in the biological sample contain a specific nucleotide sequence.

[0145] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can also include, for example, obtaining a biological sample from a subject that contains ANGPTL3 and SLC5A2 genomic nucleic acid molecules or mRNA molecules, and if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can include, 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.

[0146] In some embodiments, the assay step, detection step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of ANGPTL3 and SLC5A2 genomic nucleic acid molecules, ANGPTL3 and SLC5A2 mRNA molecules, or ANGPTL3 and SLC5A2 cDNA molecules in a biological sample that contains genetic variants compared to the corresponding ANGPTL3 and SLC5A2 reference type molecules. In some embodiments, the sequenced portion comprises one or more variants that cause loss of function (partial or complete) or are predicted to cause loss of function (partial or complete).

[0147] 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 mRNA are analyzed. In some embodiments, only the ANGPTL3 and SLC5A2 cDNA obtained from the ANGPTL3 and SLC5A2 mRNA are analyzed.

[0148] Allele-specific polymerase chain reaction techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Allele-specific primers can be used because DNA polymerase will not extend when there is a mismatch with the template.

[0149] 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 within cells obtained from a subject.

[0150] In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as an allele-specific primer or allele-specific probe, that specifically hybridizes under stringent conditions to an ANGPTL3 and SLC5A2 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and does not specifically hybridize to the corresponding ANGPTL3 and SLC5A2 reference sequence, and determining whether hybridization has occurred.

[0151] In some embodiments, the determining step, detecting step, or sequence analysis includes: a) amplifying at least a portion of the ANGPTL3 and SLC5A2 nucleic acid molecules encoding the ANGPTL3 and SLC5A2 polypeptides; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecules with a vector comprising an altered specificity probe; and d) detecting the detectable label.

[0152] In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay further includes reverse transcribing the mRNA into cDNA, for example, by reverse transcriptase polymerase chain reaction (RT-PCR).

[0153] In some embodiments, the method utilizes probes and primers having a nucleotide length sufficient to bind to a target nucleic acid sequence and specifically detect and / or discriminate polynucleotides comprising ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. The hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the selected detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. The probes and primers can have complete nucleotide sequence identity with the contiguous nucleotides within the target nucleotide sequence, but probes can be designed by conventional methods to be different from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence. 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 with the nucleotide sequence of the target nucleic acid molecule.

[0154] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, which include the use of labeled primers or probes (fluorescence in situ hybridization (FISH)) for purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule can be amplified before or simultaneously with the 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 reaction (SDA), and nucleic acid sequence-based amplification reaction (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification reaction, and thermophilic SDA (tSDA).

[0155] In hybridization techniques, stringent conditions can be employed such that a probe or primer hybridizes specifically to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater extent, such as at least 2-fold, at least 3-fold, at least 4-fold, or more (relative to background), including more than 10-fold greater (relative to background), than its hybridization to other non-target sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than its hybridization to other nucleotide sequences by at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than its hybridization to other nucleotide sequences by at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than its hybridization to other nucleotide sequences by at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than its hybridization to other nucleotide sequences by more than 10-fold (relative to background). Stringent conditions are sequence-dependent and will be different in different circumstances.

[0156] Suitable stringent conditions for promoting DNA hybridization (e.g., 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash in 2X SSC at 50°C) are known and are described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Generally, stringent conditions for hybridization and detection will be those in which the salt concentration at pH 7.0 to 8.3 is less than about 1.5 M Na + ions, typically about 0.01 to 1.0 M Na + ion concentration (or other salts), and the temperature is 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., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents (e.g., formamide). Optionally, the wash buffer can contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours. The duration of the wash time will be at least long enough to achieve equilibrium.

[0157] In some embodiments, such isolated nucleic acid molecules comprise 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 or consist thereof. In some embodiments, such isolated nucleic acid molecules comprise 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 or consist thereof. In some embodiments, the isolated nucleic acid molecule comprises at least about 18 nucleotides or consists thereof. In some embodiments, the isolated nucleic acid molecule comprises at least about 15 nucleotides or consists thereof. In some embodiments, the isolated nucleic acid molecule 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 or consist thereof. In some embodiments, the isolated nucleic acid molecule comprises about 18 to about 30 nucleotides or consists thereof. In some embodiments, the isolated nucleic acid molecule comprises at least about 15 nucleotides to at least about 35 nucleotides or consists thereof.

[0158] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to ANGPTL3 and SLC5A2 variant nucleic acid molecules, such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules. Such nucleic acid molecules can be used, for example, as probes, primers, altered specificity probes, or altered specificity primers described or exemplified herein, and include, but are not limited to, primers, probes, antisense RNAs, shRNAs, and siRNAs, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0159] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 consecutive nucleotides of a nucleic acid molecule that has 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% identity to an ANGPTL3 and SLC5A2 variant nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of from about 15 to about 100 nucleotides or from about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of from about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of from about 15 to about 35 nucleotides.

[0160] In some embodiments, the altered specificity probes and altered specificity primers comprise DNA. In some embodiments, the altered specificity probes and altered specificity primers comprise RNA.

[0161] In some embodiments, the probes and primers described herein (including altered specificity probes and altered specificity primers) have nucleotide sequences that specifically hybridize to any nucleic acid molecule disclosed herein or its complementary sequence. In some embodiments, the probes and primers specifically hybridize to any nucleic acid molecule disclosed herein under stringent conditions.

[0162] In some embodiments, primers (including altered specificity primers) can be used in next-generation sequencing or high-throughput sequencing. In some cases, primers can be modified, including altered specificity primers. In particular, primers can contain various modifications used in different steps such as massively parallel signature sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including using biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading step and the detection step. Polony sequencing is typically performed using a paired-end tag library, where the length of each DNA template molecule is about 135 bp. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adaptor can contain a 5'-biotin tag for immobilizing the DNA library onto streptavidin-coated beads.

[0163] The probes and primers described herein can be used to detect nucleotide variations within 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 a fragment thereof.

[0164] In the context of the present disclosure, "specific hybridization" means that a probe or primer (such as an altered specificity probe or an altered specificity primer) does not hybridize to the nucleic acid sequences encoding the ANGPTL3 and SLC5A2 reference genomic nucleic acid molecules, the ANGPTL3 and SLC5A2 reference mRNA molecules, and / or the ANGPTL3 and SLC5A2 reference cDNA molecules.

[0165] In some embodiments, the probe (such as an altered specificity probe) contains a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0166] The present disclosure also provides a support comprising a solid support to which any one or more of the probes disclosed herein are attached. A solid support is a solid substrate or support to which a molecule (such as any one of the probes disclosed herein) can associate. One form of the solid support is an array. Another form of the solid support is an array detector. An array detector is a solid support to which a plurality of different probes are coupled in an array, grid, or other organized pattern. One form of the solid substrate is a microtiter plate, such as a standard 96-well type. In some embodiments, a porous glass slide that typically contains one array per well can be employed.

[0167] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, the genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or orthologs from another organism (e.g., non-human mammal, rodent, mouse, or rat). It should be understood that gene sequences within a population can vary due to polymorphisms (such as single nucleotide polymorphisms).

[0168] The present disclosure also provides 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. The functional polynucleotides can act as effectors, inhibitors, regulators, and stimulators of specific activities possessed by the target molecule, or the functional polynucleotides can have entirely new activities independent of any other molecule.

[0169] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence (such as in a vector) or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be in a vector or as 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 a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where an enzyme-dependent secondary signal generation occurs. The term "label" can also refer to a "tag" or a hapten that can selectively bind to a conjugate molecule such that the conjugate molecule is used to generate a detectable signal when added together with a substrate subsequently. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag, and a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorescent substrate can be used to check for 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, an epitope tag, or the Fc portion of an immunoglobulin. Many labels include, for example, particles, fluorophores, haptens, enzymes, and their calorimetric, fluorescent, and chemiluminescent substrates and other labels.

[0170] The percent identity (or percent complementarity) between specific stretches of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the 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.), using default settings which use the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 - 489). In this context, if a percent sequence identity is mentioned, a higher percent sequence identity is preferred over a lower percent sequence identity.

[0171] The present disclosure also provides a therapeutic agent for kidney diseases for use in treating or preventing kidney diseases in a subject having ANGPTL3 and SLC5A2 variant nucleic acid molecules. Any one of the therapeutic agents for kidney diseases described herein can be used herein. Any one of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the ANGPTL3 and SLC5A2 variant nucleic acid molecules are ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules comprising any one of the genetic variants described herein, or mRNA molecules produced therefrom, or cDNA molecules produced from the mRNA molecules.

[0172] The present disclosure also provides the use of a therapeutic agent for kidney diseases for the preparation of a medicament for treating or preventing kidney diseases in a subject having ANGPTL3 and SLC5A2 variant nucleic acid molecules. Any one of the therapeutic agents for kidney diseases described herein can be used herein. Any one of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the ANGPTL3 and SLC5A2 variant nucleic acid molecules are ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules comprising any one of the genetic variants described herein, or mRNA molecules produced therefrom, or cDNA molecules produced from the mRNA molecules.

[0173] The present disclosure also provides ANGPTL3 and SLC5A2 inhibitors for use in treating or preventing kidney diseases in a subject who is of the ANGPTL3 and SLC5A2 reference type, or is heterozygous for ANGPTL3 and SLC5A2 variant nucleic acid molecules, or is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and of the reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. Any one of the ANGPTL3 and SLC5A2 inhibitors described herein can be used herein. Any one of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the ANGPTL3 and SLC5A2 variant nucleic acid molecules are ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules containing any one of the genetic variants described herein, or mRNA molecules produced therefrom, or cDNA molecules produced from the mRNA molecules.

[0174] The present disclosure also provides ANGPTL3 and SLC5A2 inhibitors for use in preparing a medicament for treating or preventing kidney diseases in a subject who is of the ANGPTL3 and SLC5A2 reference type, or is heterozygous for ANGPTL3 and SLC5A2 variant nucleic acid molecules or heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and of the reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule. Any one of the ANGPTL3 and SLC5A2 inhibitors described herein can be used herein. Any one of the ANGPTL3 and SLC5A2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the ANGPTL3 and SLC5A2 variant nucleic acid molecules are ANGPTL3 and SLC5A2 variant genomic nucleic acid molecules containing any one of the genetic variants described herein, or mRNA molecules produced therefrom, or cDNA molecules produced from the mRNA molecules.

[0175] In some embodiments, the ANGPTL3 and SLC5A2 inhibitors and the kidney disease therapeutic agent are placed in a pharmaceutical composition. In some embodiments, the ANGPTL3 and SLC5A2 inhibitors are placed in a first pharmaceutical composition and the kidney disease therapeutic agent is placed in 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 kidney disease therapeutic agent are placed in separate pharmaceutical compositions.

[0176] All patent documents, websites, other publications, accession numbers, etc., cited above or below are hereby 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. If different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of the present application is meant. The effective filing date means the earlier of the actual filing date or the filing date of the priority application for the reference accession number (if applicable). Similarly, if different versions of a publication, website, etc. are published at different times, unless otherwise indicated, the version most recently published as of the effective filing date of the present application is meant. Unless otherwise specifically stated, any feature, step, element, embodiment or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment or aspect. Although the present disclosure has been described in detail by way of 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.

[0177] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate but not limit the claimed embodiments. The following examples provide the disclosure and description to those of ordinary skill in the art of how the compounds, compositions, articles, devices, and / or methods described herein are made and evaluated, and are merely intended to be exemplary and not intended to limit the scope of any claims. Efforts have been made to ensure the accuracy of numbers (such as amounts, temperatures, etc.), but some errors and deviations may be considered. Unless otherwise indicated, parts are parts by weight, temperature is in °C or at ambient temperature, and pressure is at or near atmospheric pressure.

[0178] Example

[0179] Example 1: Loss-of-function of ANGPTL3 is associated with improved renal function and is independent of SLC5A2

[0180] To determine whether the effect of ANGPTL3 on chronic kidney disease is influenced by SGLT2 function, exome sequencing was performed on up to 677,048 participants in five studies to identify rare coding mutations. We estimated the association between eGFR and the burden of rare loss-of-function and missense variants identified in ANGPTL3 with and without adjustment for the burden of rare loss-of-function and missense variants in the gene SLC5A2 encoding SGLT2. eGFR is a widely used biomarker of renal function, where higher levels indicate better function.

[0181] In this analysis, the burden of rare (alternate 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, after adjustment for rare loss-of-function and missense variants in SLC5A2 (AAF < 0.1%), the burden of pLOF or missense genetic variants in ANGPTL3 had a similar effect on increased eGFR, indicating that the two genes interact independently of each other to increase eGFR.

[0182] Table 1: Association of the burden of rare (AAF < 0.1%) pLOF or missense variants in the ANGPTL3 gene with increased eGFR levels (outcome = eGFR)

[0183]

[0184] Abbreviations: CI, confidence interval; SD, standard deviation; mL / min, milliliters per minute; AAF, alternate allele frequency; Ref, homozygous reference genotype; Het, heterozygous carrier of rare pLOF or 5 / 5 missense variants in ANGPTL3; Hom, homozygous carrier of rare pLOF or 5 / 5 missense variants in ANGPTL3; pLOF, predicted loss-of-function; 5 / 5 missense, missense variant predicted to be deleterious by 5 out of 5 computational prediction algorithms; eGFR, estimated glomerular filtration rate.

[0185] Comparing the association of individual rare ANGPTL3 pLOF variants (AAF < 1%, excluding missense variants) with and without adjustment for SLC5A2 pLOF variants, similar increased results were observed (see Table 2), indicating that the association of rare pLOF plus missense variants reflects loss-of-function in ANGPTL3 that is independent of the effect of loss-of-function in SLC5A2.

[0186] Table 2: Association of the burden of rare (AAF < 1%) pLOF variants in the ANGPTL3 gene with increased eGFR levels (outcome = eGFR)

[0187]

[0188] Abbreviations: CI, confidence interval; SD, standard deviation; mL / min, milliliters per minute; AAF, alternate allele frequency; Ref, homozygous reference genotype; Het, heterozygous carrier of rare pLOF variants in ANGPTL3; Hom, homozygous carrier of rare pLOF variants in ANGPTL3; pLOF, predicted loss-of-function; eGFR, estimated glomerular filtration rate.

[0189] Example 2: Loss-of-function of SLC5A2 is associated with improved kidney function and is independent of ANGPTL3

[0190] Next, we compared the association of rare coding variants in SLC5A2 with increased eGFR with and without adjustment for rare coding variants in ANGPTL3. The burden of pLOF or missense genetic variants in SLC5A2 (AAF < 0.1%) had a similar effect on increased eGFR after adjustment for rare loss-of-function and missense variants in ANGPTL3 (AAF < 0.1%), indicating that these two genes act independently of each other to increase eGFR (see Table 3).

[0191] Table 3: Association of the burden of rare (AAF < 0.1%) pLOF or missense variants in the SLC5A2 gene with increased eGFR levels (Outcome = eGFR)

[0192]

[0193] Abbreviations: CI, confidence interval; SD, standard deviation; mL / min, milliliters per minute; AAF, alternate allele frequency; Ref, homozygous reference genotype; Het, heterozygous carriers of rare pLOF or 5 / 5 missense variants in SLC5A2; Hom, homozygous carriers of rare pLOF or 5 / 5 missense variants in SLC5A2; pLOF, predicted loss-of-function; 5 / 5 missense, missense variants predicted to be deleterious by 5 out of 5 computational prediction algorithms; eGFR, estimated glomerular filtration rate.

[0194] Comparing the association of individual rare SLC5A2 pLOF variants (AAF < 1%, excluding missense variants) with and without adjustment for ANGPTL3 pLOF variants, similar increased results were observed (see Table 4), indicating that the association of rare pLOF plus missense variants reflects loss-of-function in SLC5A2 that is independent of the effect of loss-of-function in ANGPTL3.

[0195] Table 4: Association of the burden of rare (AAF < 1%) pLOF variants in the SLC5A2 gene with increased eGFR levels (Outcome = eGFR)

[0196]

[0197]

[0198] Abbreviations: CI, confidence interval; SD, standard deviation; mL / min, milliliters per minute; AAF, alternative allele frequency; Ref, homozygous reference genotype; Het, heterozygous carrier of a rare pLOF variant in SLC5A2; Hom, homozygous carrier of a rare pLOF variant in SLC5A2; pLOF, predicted loss of function; eGFR, estimated glomerular filtration rate.

[0199] Example 3: Rare coding variants in ANGPTL3 and SLC5A2 are associated with improved renal function and are independent of other genes

[0200] To formally test whether the effects of rare coding variants in ANGPTL3 and SLC5A2 on eGFR are independent of each other, we performed an interaction analysis. The main effects of ANGPTL3 (i.e., the effect of the ANGPTL3 variant in individuals with the homozygous reference genotype of the SLC5A2 variant) and SLC5A2 (i.e., the effect of the SLC5A2 variant in individuals with the homozygous reference genotype of the ANGPTL3 variant) on eGFR were similar to the overall effects of the ANGPTL3 rare coding variant (see Table 1) or the SLC5A2 rare coding variant (see Table 3) on eGFR, respectively (see Table 5). The interaction terms testing whether the effect of the ANGPTL3 variant was different in SLC5A2 homozygous reference vs. SLC5A2 heterozygous variant carriers, and whether the effect of the SLC5A2 variant was different in ANGPTL3 homozygous reference vs. ANGPTL3 heterozygous variant carriers were not significant (see Table 5). This indicates that the effect of ANGPTL3 is independent of the SLC5A2 genotype status, and the effect of SLC5A2 is independent of the ANGPTL3 genotype status, and thus there is no evidence that the association between these two genotypes deviates from additivity.

[0201] Table 5: Interaction of rare (AAF < 0.1%) pLOF or missense variants in ANGPTL3 and SLC5A2 genes on eGFR (outcome = eGFR)

[0202]

[0203]

[0204] Abbreviations: CI, confidence interval; SD, standard deviation; mL / min, milliliters per minute; AAF, alternative allele frequency; Ref, homozygous reference genotype; Het, heterozygous carrier of rare pLOF or 5 / 5 missense variants in ANGPTL3 (line 1) or SLC5A2 (line 2); Hom, homozygous carrier of rare pLOF or 5 / 5 missense variants in ANGPTL3 (line 1) or SLC5A2 (line 2); pLOF, predicted loss of function; 5 / 5 missense, 5 out of 5 in silico prediction algorithms predict the missense variant as deleterious; eGFR, estimated glomerular filtration rate.

[0205] The associations between ANGPTL3, SLC5A2, and eGFR were driven by multiple rare pLOF or missense variants in the ANGPTL3 and SLC5A2 genes (see Table 6).

[0206] Table 6: Missense or pLOF variants in ANGPTL3 or SLC5A2 identified by exome sequencing and included in the gene burden association analysis

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] According to the 38th version of the human genome sequence established by the Human Genome Reference Consortium, C:P:R:A indicates the genomic coordinates of genetic variants, including chromosome (C), physical genomic position in base pairs (P), reference allele (R), and alternative allele (A). Changes in coding DNA and proteins follow the nomenclature of the Human Genome Variation Society and refer to the ANGPTL3 or SLC5A2 transcripts (ENST00000371129 or ENST00000330498, respectively) annotated in the Ensembl database (see the World Wide Web URL "useast.ensembl.org / index.html"). 'Missense (5 / 5)' and '5 / 5ms' indicate missense variants predicted to be harmful by 5 computer algorithms. pLOF indicates the predicted loss of function of the variant. The four columns on the right indicate whether the variant is included in the specified gene load grouping, with an alternative allele frequency (AAF) threshold of <1% for pLOF variants and <0.1% for pLOF or 5 / 5 missense variants.

[0237] Example 4: General method

[0238] Participating cohort

[0239] The UKB is a population-based cohort study of individuals aged between 40 and 69 years recruited through 22 assessment centres across the UK between 2006 and 2010 (Sudlow et al., PLoS Med, 2015.12, e1001779). A total of 431,835 participants from the UKB were included, along with available whole-exome sequencing and phenotype data. 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 patient cohort recruited from central and eastern Pennsylvania (USA) between 2007–2019. A total of 156,846 participants from the GHS were included, along with available whole-exome sequencing and phenotype data. The Mount Sinai Biobank Personalized Medicine cohort (SINAI) (Gottesman et al., Genet. Med., 2013, 15, 761–771) is an electronic health record-linked clinical care cohort with 25,839 individuals and available whole-exome sequencing and phenotype data. The University of Pennsylvania Medical Biobank (UPENN-PMBB) is a health system-based cohort from the Pennsylvania Biobank (Park et al., Nat. Med., 2021, 27, 66–72). The Malmö Diet and Cancer Study (MDCS) is a cohort study from Malmö, Sweden (Berglund et al., J. Intern. Med., 1993, 233, 45–51). The MCPS is a population-based prospective study from Mexico (Tapia-Conyer et al., Int. J. Epidemiol., 2006 35, 243–49).

[0240] Phenotype definition

[0241] eGFR was calculated based on clinical laboratory measurements of creatinine extracted from the electronic health records (EHRs) of participants from GHS, SINAI, and UPENN-PMBB. Median values were calculated for all participants with two or more measurements. In UKB, eGFR was calculated based on creatinine measured on a Beckman Coulter AU5800 clinical chemistry analyzer and on cystatin measured by nephelometry on a Siemens Advia 1800 clinical chemistry analyzer; both creatinine and cystatin were measured at the baseline visit of the study. In MCPS, eGFR was calculated based on creatinine measured by nuclear magnetic resonance spectroscopy and converted to clinical units as previously described (Aguilar-Ramirez et al., J. Clin. Endo. Met., 2021, 106, 2828-2839). The Chronic Kidney Disease Epidemiology Collaboration equations for creatinine and cystatin (UKB) or creatinine (all other cohorts) were used to calculate eGFR. Prior to performing genetic association analyses, eGFR values were transformed by the inverse standard normal function, applied within each ancestry group, and separately for males and females.

[0242] Genotype data

[0243] High-coverage whole-exome sequencing was performed as described previously (Science, 2021, 373:abf8683; and Nature, 2020, 586:749-756) and as summarized below. NimbleGen probes (VCRome; for a portion of the GHS cohort) or a modified version of the xGen design provided by Integrated DNA Technologies (IDT; for the remainder of GHS and other cohorts) were used for target sequence capture of the exome. Unique 6-base pair (bp) barcodes (VCRome) or 10-bp barcodes (IDT) were added to each DNA fragment during library preparation to facilitate multiplexed exome capture and sequencing. Equal amounts of samples were pooled prior to exome capture. Sequencing was performed using 75-bp paired-end reads on an Illumina v4 HiSeq 2500 (for a portion of the GHS cohort) or NovaSeq (for the remainder of GHS and other cohorts) instrument. The sequencing coverage depth (i.e., the number of sequence reads that cover each nucleotide in the genomic target region) was sufficient to provide >20x coverage of 85% of the target bases in 96% of the VCRome samples and 20x coverage of 90% of the target bases in 99% of the IDT samples. Data processing steps included sample de-multiplexing 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 assessments). 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 the snpEff software. Then, the snpEff predictions involving protein-coding transcripts with annotated start and stop were combined into a single functional impact prediction by selecting the most deleterious functional effect category for each gene. The ranks of these annotations (from most deleterious to least deleterious) were frameshift, stop-gained, stop-loss, splice acceptor, splice donor, stop-lost, in-frame insertion / deletion, missense, other annotation. Predicted LOF genetic variants included: a) insertions or deletions that result in a frameshift, b) insertions, deletions, or single nucleotide variants that result in the introduction of a premature stop codon or loss of a transcriptional start or stop site, and c) variants at donor or acceptor splice sites.Classify missense variants using computer prediction algorithms that predict harmfulness according to possible functional impacts, such as SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), 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 alternate allele frequency (AAF) and functional annotation of each variant are determined to be included in 7 gene burden exposures: 1) pLOF variants with AAF < 1%; 2) pLOF or missense variants with AAF < 1% predicted to be harmful by the 5 / 5 algorithm; 3) pLOF or missense variants with AAF < 0.1% predicted to be harmful by the 5 / 5 algorithm; 4) pLOF or missense variants with AAF < 1% predicted to be harmful by at least 1 / 5 algorithm; 5) pLOF or missense variants with AAF < 0.1% predicted to be harmful by at least 1 / 5 algorithm; 6) pLOF or any missense with AAF < 1%; 7) pLOF or any missense variant with AAF < 0.1%.

[0244] Association analysis of gene burden of rare pLOF and missense variants

[0245] We tested 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 that was adjusted for a polygenic score using REGENIE to estimate the genomic relationship matrix (Mbatchou et al., Nat. Genet., 2021, 53, 1097–1103). Stratify and adjust the analysis for age, age 2 squared, sex, age-by-sex and age-by-sex 2 interaction terms, experiment batch-related covariates, 10 principal components from common variant sources, and 20 principal components from rare variant sources. Use fixed-effect inverse-variance weighted meta-analysis to combine cross-cohort results of each variant-phenotype association. In the gene burden test, if all individuals carry one or more eligible rare variants (as described above, based on frequency and functional annotation), all individuals are labeled as heterozygotes, and if they carry any eligible variant in the homozygous state, they are labeled as homozygotes. Then use this "compound genotype" to test the association. Further analyze the burden exposure with the strongest association with eGFR and the burden of pLOF variants with AAF < 1%.

[0246] To examine whether the effect of ANGPTL3 gene burden on eGFR varies with SLC5A2 gene burden and vice versa, we used two approaches. First, we adjusted for the second gene burden by including it as a covariate in the linear regression model between the first gene burden and eGFR. We compared these results with those of the 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 fitted with interaction terms for ANGPTL3 gene burden, SLC5A2 gene burden, and ANGPTL3*SLC5A2 gene burden. The p-value of the interaction term allowed assessment of whether the effect of ANGPTL3 gene burden was significantly different between different genotypes of SLC5A2 gene burden (and vice versa). The two analyses were performed as described in the previous paragraph, substituting the modified REGENIE polygenic score excluding chromosomes containing the ANGPTL3 and SLC5A2 genes.

[0247] In addition to the modifications described herein, various modifications of the described subject matter will be apparent to those of ordinary skill 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 hereby incorporated by reference in its entirety and for all purposes.

Claims

1. A method of treating a subject having a kidney disease or at risk of developing a kidney disease, the method comprising administering to the subject an angiopoietin-like 3 (ANGPTL3) inhibitor and a solute carrier family 5 member 2 (SLC5A2) inhibitor.

2. The method according to claim 1, wherein the kidney disease is chronic kidney disease.

3. The method according to claim 1, wherein the kidney disease is diabetic kidney disease.

4. The method according to claim 1, wherein the kidney disease is chronic glomerulonephritis.

5. The method according to claim 1, wherein the kidney disease is nephronophthisis.

6. The method according to claim 1, wherein the kidney disease is chronic interstitial nephritis.

7. The method according to claim 1, wherein the kidney disease is nephrosclerosis.

8. The method according to any one of claims 1 to 7, wherein the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ANGPTL3 nucleic acid molecule.

9. The method according to claim 8, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

10. The method according to claim 9, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

11. The method according to claim 9, wherein the inhibitory nucleic acid molecule comprises siRNA.

12. The method according to any one of claims 1 to 7, wherein the ANGPTL3 inhibitor comprises a small molecule.

13. The method according to any one of claims 1 to 7, wherein the ANGPTL3 inhibitor comprises an antibody.

14. The method according to claim 13, wherein the antibody comprises eculizumab.

15. The method according to any one of claims 1 to 14, wherein the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an SLC5A2 nucleic acid molecule.

16. The method according to claim 15, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

17. The method according to claim 16, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

18. The method according to claim 16, wherein the inhibitory nucleic acid molecule comprises siRNA.

19. The method according to any one of claims 1 to 14, wherein the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof.

20. The method according to any one of claims 1 to 19, further comprising detecting the presence or absence of an ANGPTL3 variant nucleic acid molecule in a biological sample from the subject, and detecting the presence or absence of an SLC5A2 variant nucleic acid molecule.

21. The method according to claim 20, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide.

22. The method according to claim 20 or claim 21, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

23. The method according to any one of claims 20 to 22, further comprising administering a renal disease therapeutic agent to a subject who is: i) homozygous for the ANGPTL3 reference type or the ANGPTL3 variant nucleic acid molecule, and ii) homozygous for the SLC5A2 reference type or the SLC5A2 variant nucleic acid molecule.

24. A method of treating a subject with a renal disease therapeutic agent by administering the renal disease therapeutic agent, wherein the subject has a renal disease or is at risk of developing a renal disease, the method comprising: determining whether the subject has an angiopoietin-like 3 (ANGPTL3) variant nucleic acid molecule and whether the subject has a solute carrier family 5 member 2 (SLC5A2) variant nucleic acid molecule by the following steps: 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: i) the ANGPTL3 variant nucleic acid molecule, and ii) the SLC5A2 variant nucleic acid molecule; and administering or continuing to administer the renal disease therapeutic agent to a subject who is homozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule; or administering or continuing to administer the renal disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor to a subject who is: i) heterozygous for the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, ii) heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and homozygous for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, or iii) homozygous for the ANGPTL3 reference type and the SLC5A2 reference type; wherein the presence of the genotype having the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule indicates a reduced risk of the subject developing a renal disease.

25. The method according to claim 24, wherein the renal disease is chronic kidney disease.

26. The method according to claim 24, wherein the renal disease is diabetic kidney disease.

27. The method according to claim 24, wherein the renal disease is chronic glomerulonephritis.

28. The method according to claim 24, wherein the kidney disease is nephronophthisis.

29. The method according to claim 24, wherein the kidney disease is chronic interstitial nephritis.

30. The method according to claim 24, wherein the kidney disease is nephrosclerosis.

31. The method according to any one of claims 24 to 30, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide.

32. The method according to any one of claims 24 to 31, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

33. The method according to any one of claims 24 to 32, wherein the subject is: i) heterozygous for the ANGPTL3 reference type or the ANGPTL3 variant nucleic acid molecule, and ii) heterozygous for the SLC5A2 reference type or the SLC5A2 variant nucleic acid molecule, and the kidney disease therapeutic agent and / or the ANGPTL3 inhibitor and the SLC5A2 inhibitor are administered or continued to be administered to the subject.

34. The method according to any one of claims 24 to 33, wherein the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to the ANGPTL3 nucleic acid molecule.

35. The method according to claim 34, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

36. The method according to claim 35, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

37. The method according to claim 35, wherein the inhibitory nucleic acid molecule comprises siRNA.

38. The method according to any one of claims 24 to 33, wherein the ANGPTL3 inhibitor comprises a small molecule.

39. The method according to any one of claims 24 to 33, wherein the ANGPTL3 inhibitor comprises an antibody.

40. The method according to claim 39, wherein the antibody comprises eculizumab.

41. The method according to any one of claims 24 to 40, wherein the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to the SLC5A2 nucleic acid molecule.

42. The method according to claim 41, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

43. The method according to claim 42, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

44. The method according to claim 42, wherein the inhibitory nucleic acid molecule comprises siRNA.

45. The method according to any one of claims 20 to 40, wherein the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof.

46. A method for identifying a subject at increased risk of developing a kidney disease, the 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 in the biological sample; wherein: when the subject is ANGPTL3 reference type and SLC5A2 reference type, then the subject has an increased risk of developing a kidney disease; and when 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 when the subject is heterozygous for one of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule and reference type for the other of the ANGPTL3 variant nucleic acid molecule and the SLC5A2 variant nucleic acid molecule, then the subject has a reduced risk of developing a kidney disease.

47. The method according to claim 46, wherein the kidney disease is chronic kidney disease.

48. The method according to claim 46, wherein the kidney disease is diabetic kidney disease.

49. The method according to claim 46, wherein the kidney disease is chronic glomerulonephritis.

50. The method according to claim 46, wherein the kidney disease is nephronophthisis.

51. The method according to claim 46, wherein the kidney disease is chronic interstitial nephritis.

52. The method according to claim 46, wherein the kidney disease is renal sclerosis.

53. The method according to any one of claims 46 to 52, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide.

54. The method according to any one of claims 46 to 53, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

55. The method according to any one of claims 46 to 54, further comprising administering to the subject a kidney disease therapeutic agent and / or an ANGPTL3 inhibitor and an SLC5A2 inhibitor, the subject being: i) ANGPTL3 reference type or heterozygous for the ANGPTL3 variant nucleic acid molecule, and ii) SLC5A2 reference type or heterozygous for the SLC5A2 variant nucleic acid molecule.

56. The method according to claim 55, wherein the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ANGPTL3 nucleic acid molecule.

57. The method according to claim 56, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA).

58. The method according to claim 57, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

59. The method according to claim 57, wherein the inhibitory nucleic acid molecule comprises siRNA.

60. The method according to claim 55, wherein the ANGPTL3 inhibitor comprises a small molecule.

61. The method according to claim 55, wherein the ANGPTL3 inhibitor comprises an antibody.

62. The method according to claim 61, wherein the antibody comprises eculizumab.

63. The method according to any one of claims 55 to 62, wherein the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an SLC5A2 nucleic acid molecule.

64. The method according to claim 63, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA).

65. The method according to claim 63, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

66. The method according to claim 63, wherein the inhibitory nucleic acid molecule comprises siRNA.

67. The method according to any one of claims 55 to 62, wherein the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin, or tofogliflozin, or any combination thereof.

68. A therapeutic agent for kidney diseases for use in treating or preventing kidney diseases in a subject having an ANGPTL3 variant nucleic acid molecule and an SLC5A2 variant nucleic acid molecule.

69. The method according to claim 68, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, splice site variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant, in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide.

70. The method according to claim 68 or claim 69, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, splice site variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant, in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

71. An ANGPTL3 inhibitor and an SLC5A2 inhibitor for use in treating or preventing kidney diseases in a subject who is: i) heterozygous for an ANGPTL3 reference type or an ANGPTL3 variant nucleic acid molecule, and ii) heterozygous for an SLC5A2 reference type or an SLC5A2 variant nucleic acid molecule.

72. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 71, wherein the ANGPTL3 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated ANGPTL3 polypeptide.

73. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 71 or claim 72, wherein the SLC5A2 variant nucleic acid molecule is a missense variant, a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated SLC5A2 polypeptide.

74. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to any one of claims 71 to 73, wherein the ANGPTL3 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ANGPTL3 nucleic acid molecule.

75. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 74, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

76. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 75, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

77. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 75, wherein the inhibitory nucleic acid molecule comprises siRNA.

78. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to any one of claims 71 to 73, wherein the ANGPTL3 inhibitor comprises a small molecule.

79. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to any one of claims 71 to 73, wherein the ANGPTL3 inhibitor comprises an antibody.

80. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 79, wherein the antibody comprises evinacumab.

81. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to any one of claims 71 to 80, wherein the SLC5A2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an SLC5A2 nucleic acid molecule.

82. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 81, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

83. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 82, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

84. The ANGPTL3 inhibitor and SLC5A2 inhibitor according to claim 82, wherein the inhibitory nucleic acid molecule comprises siRNA. The ANGPTL3 inhibitor and the SLC5A2 inhibitor according to any one of claims 71 to 80, wherein the SLC5A2 inhibitor comprises canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, luseogliflozin or tofogliflozin or any combination thereof.

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