Renal dysfunction markers as biomarkers for acute hepatic porphyria (AHP)

Abstract

The present disclosure provides biomarkers for the diagnosis and monitoring of acute hepatic porphyria (AHP). The present disclosure further provides methods for selecting drugs for the treatment of AHP using the biomarkers. The present disclosure further provides kits for practicing the methods provided herein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 252,919, filed October 6, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing XML file that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. Said XML copy, created on September 29, 2022, is named 121301-18820_SeqListing.xml and is 75,394 bytes in size.

[0003] Field of the Disclosure This disclosure relates to biomarkers for acute hepatic porphyria (AHP). More specifically, this disclosure relates to renal injury biomarkers as biomarkers for AHP. [Background technology]

[0004] Acute hepatic porphyrias (AHP) are a group of rare genetic disorders caused by deficiencies of enzymes in the heme biosynthetic pathway. AHP has four subtypes: acute intermittent porphyria (AIP), variegate porphyria (VP), hereditary coproporphyria (HCP), and ALAD deficiency porphyria (ADP). The most common subtype is AIP.

[0005] In patients with AHP, accumulation of the heme pathway intermediates delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) leads to acute attacks and long-term complications, including hypertension and chronic kidney disease, which are present in 30-60% of patients with biochemically active AIP. Summary of the Invention

[0006] According to one embodiment, a method of treating a human subject having acute hepatic porphyria (AHP) is provided, the method comprising, for example, administering to the subject a therapeutic agent that reduces expression of 5'-aminolevulinic acid synthase 1 (ALAS1) in response to determining a level of a biomarker (e.g., a renal injury biomarker) that is elevated in the subject compared to a reference level, thereby treating the subject.

[0007] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0008] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0009] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0010] In some embodiments, the subject is a chronic high excreter (CHE). In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0011] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0012] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0013] According to another aspect, a method of treating a human subject having or at risk of having AHP is provided, the method including, for example, obtaining or having obtained a biological sample from a subject, performing or having performed an assay to determine a level of a biomarker (e.g., a renal impairment biomarker) in the biological sample, and administering to the subject a therapeutic agent that reduces expression of ALAS1 if the subject has an elevated level of the biomarker (e.g., a renal impairment biomarker) compared to a reference value.

[0014] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0015] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0016] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0017] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0018] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0019] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0020] According to another aspect, a method of treating a human subject having or at risk of having AHP is provided, which may include providing a therapeutic agent that reduces expression of ALAS1, detecting an elevated level of a biomarker (e.g., a renal impairment biomarker) in the subject compared to a reference level, and administering the therapeutic agent to the subject if the level of the biomarker (e.g., a renal impairment biomarker) is higher than the reference level.

[0021] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0022] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0023] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0024] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0025] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0026] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0027] According to another aspect, a therapeutic agent that reduces expression of ALAS1 is provided for use in treating a human subject having AHP, wherein the subject has an elevated level of a biomarker (e.g., a renal injury biomarker) compared to a reference level.

[0028] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0029] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0030] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0031] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0032] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0033] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0034] According to another aspect, a therapeutic agent that reduces expression of ALAS1 is provided for use in a method of treating a human subject having AHP, the method comprising determining whether the patient has an elevated level of a biomarker (e.g., a renal impairment biomarker) compared to a reference level of the renal impairment biomarker.

[0035] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0036] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0037] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0038] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0039] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0040] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0041] According to another aspect, an in vitro method for diagnosing AHP in a subject is provided, which may, for example, include: (a) determining a level of a biomarker (e.g., a renal impairment biomarker) in a sample from the subject; (b) comparing the level of the biomarker (e.g., a renal impairment biomarker) determined in step (a) with a reference level of the biomarker (e.g., a renal impairment biomarker); and (c) assessing whether the subject suffers from AHP, wherein an increase in the level of the biomarker (e.g., a renal impairment biomarker) determined in step (a) compared to the reference level of the biomarker (e.g., a renal impairment biomarker) indicates that the subject suffers from AHP.

[0042] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0043] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0044] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0045] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0046] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0047] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0048] According to another aspect, there is provided a therapeutic agent that reduces expression of ALAS1 for use in treating AHP in a subject identified as suffering from AHP using the methods defined herein.

[0049] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0050] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0051] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0052] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0053] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0054] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker).

[0055] According to another aspect, a therapeutic agent that reduces the expression of ALAS1 is provided for use in a method for treating AHP. The method may, for example, include: (a) determining the level of a biomarker (e.g., a renal impairment biomarker) in a sample from a subject; (b) comparing the level of the biomarker (e.g., a renal impairment biomarker) determined in step (a) with a reference level of the biomarker (e.g., a renal impairment biomarker); (c) assessing whether the subject suffers from AHP, where an increase in the level of the biomarker (e.g., a renal impairment biomarker) determined in step (a) compared to the reference level of the biomarker (e.g., a renal impairment biomarker) indicates that the subject suffers from AHP; and (d) administering a therapeutic agent that reduces the expression of ALAS1 to the subject identified in step (c) as suffering from AHP.

[0056] In some embodiments, the biomarkers are selected from one or more (eg, two, three, four, five, or all) of: KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1.

[0057] In some embodiments, the biomarker is a renal injury biomarker. In some embodiments, the renal injury biomarker is selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0058] In some embodiments, the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid drug. In some embodiments, the nucleic acid drug is an RNAi agent or an antisense oligonucleotide. In some embodiments, the nucleic acid drug is an RNAi agent described herein. In some embodiments, the nucleic acid drug is givosiran.

[0059] In some embodiments, the subject is a chronic high excretor. In some embodiments, the subject has recurrent acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has elevated levels of ALA and / or PBG. In some embodiments, the subject has a mutation associated with AHP. In some embodiments, the subject does not have a mutation associated with AHP. In some embodiments, the subject has a history of renal dysfunction. In some embodiments, the subject has been diagnosed with renal impairment. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject further suffers from one or more symptoms associated with AHP.

[0060] In some embodiments, the reference level is the level of a healthy control or the previous level of the same subject.In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) is increased at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10-fold) compared to the reference level of the biomarker (e.g., a renal impairment biomarker).In some embodiments, the level is determined in a sample selected from the subject's blood, plasma, serum, urine, or feces.

[0061] In some embodiments, the subject is being treated with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. In some embodiments, the method further comprises discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker (e.g., a renal impairment biomarker). [Brief description of the drawings]

[0062] [Figure 1] 1A-1B contain graphs showing the protein expression differences of plasma proteins between two cohorts of AHP patients and healthy controls. [Figure 2-1] Figures 2A-2C include graphs showing kidney injury molecule-1 (KIM1) levels in different populations studied, KIM1 levels in patients with a history of renal dysfunction, and KIM1 levels plotted against estimated glomerular filtration rate (eGFR). [Figure 2-2] Same as above. [Figure 3-1] 3A-3C include graphs showing matrix metalloproteinase-7 (MMP7) levels in different populations studied, MMP7 levels in patients with a history of renal dysfunction, and MMP7 levels plotted against eGFR. [Figure 3-2] Same as above. [Figure 4-1] 4A-4C include graphs showing neutrophil gelatinase-associated lipocalin (NGAL) levels in the different populations studied, NGAL levels in patients with a history of renal dysfunction, and NGAL levels plotted against eGFR. [Figure 4-2] Same as above. [Figure 5-1] 5A-5C include graphs showing cystatin 3 (CST3) levels in the different populations studied, CST3 levels in patients with a history of renal dysfunction, and CST3 levels plotted against eGFR. [Figure 5-2] Same as above. [Figure 6-1]Figures 6A-6C include graphs showing chitinase-3-like protein 1 (CHI3L1) levels in different populations studied, CHI3L1 levels in patients with a history of renal dysfunction, and CHI3L1 levels plotted against eGFR. [Figure 6-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Hereditary porphyrias are a family of disorders caused by deficiencies in the activity of certain enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway. Deficiencies in the enzymes of the porphyrin pathway result in insufficient heme production and in the accumulation of porphyrin precursors and porphyrins, which are toxic to tissues in high concentrations.

[0064] Among the hereditary porphyrias, acute intermittent porphyria (AIP, e.g., autosomal dominant AIP), variegate porphyria (VP, e.g., autosomal dominant VP), hereditary coproporphyria (HCP, e.g., autosomal dominant HCP), and 5'-aminolevulinic acid (also known as δ-aminolevulinic acid or ALA) dehydratase deficiency porphyria (ADP, e.g., autosomal recessive ADP) are classified as acute hepatic porphyrias and present with acute neurological attacks that can be life-threatening. The acute attacks are characterized by autonomic, peripheral, and central nervous symptoms, including severe abdominal pain, hypertension, tachycardia, constipation, motor weakness, paralysis, and seizures. If not treated appropriately, quadriplegia, respiratory failure, and death can result. Various factors, including cytochrome P450-inducing drugs, diet, and hormonal changes, induce acute attacks by increasing the activity of hepatic 5'-aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme in the heme biosynthetic pathway. In acute porphyrias, such as AIP, VP, HCP, and ADP, the deficiency of the respective enzymes can lead to the production and accumulation of one or more neurotoxic substrates (e.g., porphyrins and / or porphyrin precursors, such as ALA and / or PBG) in the liver and the occurrence of acute attacks. (Balwani, M and Desnick, RJ, Blood, 120:4496-4504, 2012).

[0065] AIP, also called porphobilinogen deamino acidase (PBGD) deficiency or hydroxymethylbilane synthase (HMBS) deficiency, is the most common acute hepatic porphyria. The prevalence of AIP is estimated at 5-10 per 100,000, with approximately 5-10% of patients being symptomatic. AIP is an autosomal dominant disorder caused by mutations in the HMBS gene that reduce the activity of the HMBS enzyme to, for example, half of normal activity.

[0066] Currently, the diagnosis of this disease is difficult. Diagnosis of porphyria may involve evaluation of family history, evaluation of levels of porphyrin precursors in urine, blood or feces, and / or evaluation of enzyme activity and DNA mutation analysis. Differential diagnosis of porphyria may involve determining the type of porphyria by measuring (e.g., by chromatography and fluorometry) the individual levels of porphyrins or porphyrin precursors (e.g., ALA, PBG) in urine, feces, and / or plasma. Generally, however, samples must be obtained during an attack. The diagnosis of AIP may be confirmed by demonstrating that PBG deaminoase activity in red blood cells is 50% or less of normal levels. Testing for DNA mutations may be another way to diagnose AIP.

[0067] AHP may present with recurrent acute attacks or in the form of chronic hyperexcretor (CHE). CHE is a group of AHP patients who have genetic mutations and elevated ALA and PBG levels but do not experience acute attacks. Diagnosis of CHE is more difficult. DNA mutation testing may be required for patients and at-risk family members. Thus, the diagnosis of AHP, and especially AIP, typically requires confirmation by DNA testing and identification of specific pathogenic gene mutations (e.g., mutations in HMBS).

[0068] Treatment of acute attacks typically requires hospitalization for control and treatment of acute symptoms, including, for example, abdominal pain, seizures, dehydration / hyponatremia, nausea / vomiting, tachycardia / hypertension, and urinary retention / ileus. For example, abdominal pain may be treated, for example, with narcotic analgesics, seizures may be treated with seizure prophylaxis and possibly with medication (although many antiepileptic drugs are contraindicated), nausea / vomiting may be treated, for example, with phenothiazines, and tachycardia / hypertension may be treated, for example, with beta-blockers. Treatment may include withdrawal of unsafe medications, monitoring of respiratory function as well as muscle strength and neurological status. Hemin is increasingly being given, but mild attacks (e.g., attacks without paralysis or hyponatremia) may be treated with at least 300 g of intravenous 10% glucose per day. Acute attacks are typically treated with intravenous hemin (3-4 mg / kg daily for 4-14 days) as soon as possible, and with IV glucose while waiting for the IV hemin to take effect. Clinical improvement is usually seen within 3-4 days of initiating hemin, with reduced levels of ALA and PBG.

[0069] Hemin (PANHEMATIN® or injectable hemin, formerly known as hematin) is one of the current treatments for acute neurological attacks. PANHEMATIN® is protoporphyrin IX derived from processed red blood cells (PRBCs) and containing a ferric ion (heme B) with a chloride ligand. Heme acts to limit the synthesis of porphyrins in the liver and / or bone marrow. The exact mechanism by which hemin brings about improvement in patients with acute symptoms of hepatic porphyria is unclear, but it is thought to work through (feedback) inhibition of delta-aminolevulinic acid (ALA) synthase, the rate-limiting enzyme in the porphyrin / heme biosynthetic pathway. PANHEMATIN® is thought to provide exogenous heme for feedback inhibition of ALAS1, thereby decreasing the production of ALA and PBG. Patients generally respond well, but the effect of normalizing their urinary ALA and PBG concentrations to normal levels is relatively slow. Intravenous hemin is rapidly metabolized, so three to four infusions are usually required to effectively treat or prevent an acute attack.

[0070] Givosiran (GIVLAARI®) is an AHP treatment that targets ALAS1 mRNA, causing its degradation and reducing production of the neurotoxic intermediates ALA and PBG. Clinical trials of GIVLAARI® have demonstrated rapid and sustained reduction of ALA and PBG.

[0071] Delay in administration of therapeutic drugs or continued exposure to exacerbating factors may result in more severe complications, including motor neuronopathy and associated symptoms (e.g., weakness, paralysis). In severe cases, respiratory failure and paralysis may occur. Recovery from neurological symptoms may take a significant amount of time to resolve. Therefore, rapid and accurate detection and diagnosis can reduce the occurrence of severe complications in AHP patients.

[0072] To gain further insight into AHP and identify proteomic changes associated with AHP, plasma proteomic analysis was performed in patients with AHP, including those experiencing recurrent acute attacks and chronic high excretors (CHE), to understand the proteome in more detail. This study identified plasma biomarkers associated with AHP that can be used to provide a less invasive measure, thereby facilitating faster diagnosis and improved therapeutic intervention in patients.

[0073] In the analysis, 1196 unique proteins were measured across samples from multiple cohorts. Plasma levels of 212 proteins were found to be significantly different between healthy controls and AHP patients. The most prominent proteins include amyloid-like protein 1 (APLP1), kidney injury molecule 1 (KIM1), and matrix metalloproteinase 7 (MMP7) (Figures 1A-1B). Two of the most prominent proteins, KIM1 and MMP7, are markers of renal injury in several settings. At baseline, AHP patients have significantly elevated levels of APLP1, KIM1, and MMP7 compared to healthy controls. Based on the plasma proteomic analysis and characterization of plasma levels in renal injury, other renal injury biomarkers were explored. Three additional markers of kidney injury, neutrophil gelatinase-associated lipocalin (NGAL), cystatin 3 (CST3), and chitinase 3-like protein 1 (CHI3L1), showed significant increases in patients with AHP.

[0074] One of the challenges in treating AHP is the delay in obtaining a proper diagnosis. Now that there is an effective treatment for AHP, there is an increased urgency in quickly identifying and treating subjects with AHP. Although DNA may be conveniently collected and analyzed by medical personnel, the time and skill required to perform an assessment of pathogenic gene mutations for AHP is not within the scope of a typical practitioner in routine care. The present disclosure provides protein biomarkers, including renal damage biomarkers, for assessing subjects with or at risk of having AHP, such as subjects with elevated levels of ALA and / or PBG, or subjects with mutations in AHP-associated genes. Early detection of elevated levels of biomarkers (e.g., renal damage biomarkers) can be used to rapidly initiate treatment of subjects with drugs that reduce expression of ALAS1 and treat AHP before overt symptoms develop.

[0075] The present disclosure further provides a method of selecting a therapeutic agent for treating a subject with AHP based on the level of a biomarker (e.g., a renal injury biomarker), including, but not limited to, APLP1, KIM1, MMP7, NGAL, CST3, and / or CHI3L1. Drugs that reduce the expression of ALAS1 have been proven effective in treating AHP, including subjects experiencing recurrent acute attacks and CHE, in pivotal trials.

[0076] The present disclosure further provides diagnostic kits for detection of biomarkers (e.g., renal injury biomarkers), including but not limited to APLP1, KIM1, MMP7, NGAL, CST3, and / or CHI3L1, for use in the methods disclosed herein.

[0077] definition In order that this disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that wherever a value or range of values ​​for a parameter is described, values ​​and ranges between the described values ​​are also intended to be within the scope of the disclosure.

[0078] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0079] As used herein, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to." As used herein, the term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0080] The term "about" is used herein to mean within the typical tolerance range in the art. For example, "about" can be understood as up to about two standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is listed before a number of values ​​or ranges, it is understood that "about" can modify each value of the number of values ​​or ranges.

[0081] The term "at least" preceding a value or values ​​is understood to include the value adjacent to the term "at least" and all subsequent values, or all integers that may be logically included as is apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least precedes a plurality of values ​​or ranges, it is understood that "at least" may modify each value of the plurality of values ​​or ranges.

[0082] As used herein, "less than" or "less than" is understood to refer to the value adjacent to the phrase and to a value from an appropriate lower value or integer, as appropriate in the context, down to 0. For example, a duplex having an overhang of "2 nucleotides or less" will have an overhang of 2, 1, or 0 nucleotides. When "less than" is listed before a plurality of values ​​or ranges, it is understood that "less than" can modify each value of the plurality of values ​​or ranges.

[0083] As used herein, "or" is to be understood as "and / or" unless the context indicates otherwise.

[0084] As used herein, a method of detection may include determining that the amount of the analyte is below the level of detection of the method.

[0085] As used herein, an "AHP therapeutic" is understood as a therapeutic that alleviates one or more symptoms of AHP. An AHP therapeutic can be, for example, a therapeutic that reduces expression of ALAS1 or a therapeutic that stabilizes ALAS1. In some embodiments, an AHP therapeutic prevents the production and accumulation of ALA and / or PBG.

[0086] As used herein, a "therapeutic agent that reduces the expression of ALAS1" or the like is understood as a therapeutic agent that reduces the levels of ALAS1 RNA, ALAS1 protein, or both ALAS1 RNA and ALAS1 protein. In some embodiments, a therapeutic agent that reduces the expression of ALAS1 is a therapeutic agent that promotes the degradation of mRNA encoding ALAS1, or a therapeutic agent that inhibits the translation of mRNA encoding ALAS1. Such agents include, but are not limited to, nucleic acid medicines, such as, for example, RNAi interference agents and antisense oligonucleotide drugs. Such agents can typically inhibit the expression of both wild-type and mutant ALAS1. The amount of ALAS1 in the subject is reduced, thereby reducing the production and accumulation of ALA and / or PBG. In some embodiments, the therapeutic agent is an iRNA.

[0087] As used herein, a "renal injury biomarker" or "kidney injury biomarker" is understood to be at least a fragment of a human polypeptide sequence that has previously been associated with renal injury or kidney damage. For example, the injury may be chronic renal injury or chronic kidney failure, or acute renal injury or acute kidney injury.

[0088] As used herein, "kidney injury molecule 1" or "KIM1" is understood to mean at least one fragment of the human KIM1 polypeptide sequence, e.g., accession numbers NP_036338.2 (SEQ ID NO: 1), NP_001166864.1 (SEQ ID NO: 2), or NP_001295085.1 (SEQ ID NO: 3). In some embodiments, KIM1 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification methods, such as ELISA assays or immunoblotting; chromatographic methods; or single molecule arrays (SIMOA).

[0089] As used herein, "matrix metalloprotease 7" or "MMP7" is understood to refer to at least one fragment of the human MMP7 polypeptide sequence, e.g., accession number NP_002414.1 (SEQ ID NO: 4). In some embodiments, MMP7 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification methods, e.g., ELISA assays or immunoblotting; chromatographic methods; or single molecule arrays (SIMOA).

[0090] As used herein, "neutrophil gelatinase-associated lipocalin" or "NGAL" is understood to refer to at least a fragment of the human NGAL polypeptide sequence, e.g., accession number NP_005555.2 (SEQ ID NO: 5). In some embodiments, NGAL can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification methods, e.g., ELISA assays or immunoblotting; chromatographic methods; or single molecule arrays (SIMOA).

[0091] As used herein, "cystatin 3" or "CST3" is understood as at least one fragment of the human CST3 polypeptide sequence, e.g., accession numbers NP_000090.1 (SEQ ID NO: 6) or NP_001275543.1 (SEQ ID NO: 7). In some embodiments, CST3 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification methods, e.g., ELISA assays or immunoblotting; chromatographic methods; or single molecule arrays (SIMOA).

[0092] As used herein, "chitinase 3-like protein 1" or "CHI3L1" is understood to mean at least a fragment of the human CHI3L1 polypeptide sequence, such as accession number NP_001267.2 (SEQ ID NO: 8). In some embodiments, CHI3L1 can be specifically identified by any clinically acceptable diagnostic method, such as an antibody-based identification method, such as an ELISA assay or immunoblotting; a chromatographic method; or a single molecule array (SIMOA).

[0093] As used herein, "amyloid-like protein 1" or "APLP1" is understood to mean at least one fragment of the human APLP1 polypeptide sequence, e.g., accession numbers NP_005157.1 (SEQ ID NO: 9) or NP_001019978.1 (SEQ ID NO: 10). In some embodiments, APLP1 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification methods, e.g., ELISA assays or immunoblotting; chromatographic methods; or single molecule arrays (SIMOA).

[0094] A "reference level" as used herein is understood as a predefined level to which a level obtained from an assay, e.g., a level of a biomarker, e.g., a level of a protein biomarker, is compared. In certain embodiments, the reference level may be a control level determined for a population of healthy individuals, e.g., a population that does not have a disease or condition associated with an altered level of the biomarker, and does not have a predisposition, e.g., a genetic predisposition, for a disease or condition associated with an altered level of the biomarker. In certain embodiments, the population should be matched for certain criteria, e.g., age, sex, etc. In certain embodiments, the reference level of a biomarker is a previous level of the same subject, e.g., a level before the onset of symptomatic disease or before the initiation of treatment. Typically, samples are obtained from a subject at clinically relevant intervals, e.g., at least 3 months apart, at least 6 months apart, or at least 9 months apart, such that the change in the biomarker can be observed. It is understood that if more than two samples are obtained from one subject, any of the previous samples can serve as a reference level.

[0095] As used herein, "change compared to a reference level" and the like is understood as a statistically or clinically significant change in the level of a biomarker, e.g., the change in the level of a protein biomarker compared to the reference level is greater than the typical standard deviation of the assay procedure. Furthermore, the change should be clinically relevant. The change compared to the reference level may be determined as a percentage change. For example, if the reference level for biomarker X is 100 pg / ml and the level of biomarker X in a subject is 150 pg / ml, then ((150 pg / ml-100 pg / ml) / 100 pg / ml)X100%=50%, and the level is increased by 50%. If the level of biomarker X in a subject is 300 pg / ml, the level is increased by 300%. If the level of biomarker X in a subject is 50 pg / ml, the level is decreased by 50%. In certain embodiments, the change compared to the reference level is increased by at least 50%. In certain embodiments, the change compared to the reference level is increased by at least 100%, at least 200%, or at least 300%. In certain embodiments, the change compared to the reference sample is decreased by at least 25%. In certain embodiments, the change compared to the reference sample is decreased by at least 50%.

[0096] As used herein, a "subject biological sample" or a "subject sample" includes one or more bodily fluids, cells, or tissues isolated from a subject. Examples of biological fluids include blood, serum, serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, feces, saliva, and the like. Tissue samples may include samples of tissues, organs, or localized regions. For example, samples may be derived from specific organs, organ sites, or bodily fluids or cells within those organs. In certain embodiments, the sample may be liver tissue or may be derived from the liver. In some embodiments, a "subject biological sample" may refer to the subject's blood, or serum or plasma derived from blood. In some embodiments, the bodily fluid is substantially free of cells, e.g., free of cells.

[0097] As used herein, "administering a therapeutic agent" is understood as providing a therapeutic agent to a subject. In embodiments, a therapeutic agent is provided in an appropriate dosage and route of administration for that therapeutic agent, e.g., as provided by the labeling of the therapeutic agent.

[0098] As used herein, "ALAS1" (also known as ALAS-1; delta-aminolevulinic acid synthase 1; delta-ALA synthase 1; 5'-aminolevulinic acid synthase 1; ALAS-H; ALASH; ALAS-N; ALAS3; EC 2.3.1.37; non-specific mitochondrial 5-aminolevulinic acid synthase; ALAS; MIG4; OTTHUMP00000212619; OTTHUMP00000212620; OTTHUMP00000212621; OTTHUMP00000212622; migration-inducing protein 4; EC 2.3.1) refers to a nuclear-encoded mitochondrial enzyme that is the first and typically rate-limiting enzyme in the mammalian heme biosynthetic pathway. ALAS1 catalyzes the condensation of glycine with succinyl-CoA to form delta-aminolevulinic acid (ALA). The human ALAS1 gene is ubiquitously expressed, resides on chromosome 3p21.1, and typically encodes a sequence of 640 amino acids. In contrast, the ALAS-2 gene, which encodes an isozyme, is expressed only in erythrocytes, resides on chromosome Xp11.21, and typically encodes a sequence of 550 amino acids.

[0099] As used herein, "ALAS1 protein" refers to any protein variant of ALAS1 of any species (e.g., human, mouse, non-human primate), as well as any mutants and fragments thereof that maintain ALAS1 activity. Similarly, "ALAS1 transcript" refers to any transcript variant of ALAS1 of any species (e.g., human, mouse, non-human primate). The sequence of the human ALAS1 mRNA transcript can be found, for example, in NM_000688.4 (SEQ ID NO: 11). The level of the encoded mature ALAS1 protein is regulated by heme: in mitochondria, high heme levels downregulate the mature enzyme, while low heme levels upregulate the mature enzyme. Multiple alternative splicing variants have been identified that code for the same protein.

[0100] The terms "iRNA", "RNAi", "iRNA agent", or "RNAi agent" as used herein refer to an agent that contains RNA as that term is defined herein and that mediates targeted cleavage of an RNA transcript, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the iRNA described herein results in inhibition of ALAS1 expression. Inhibition of ALAS1 expression may be assessed based on a reduction in the level of ALAS1 mRNA or a reduction in the level of ALAS1 protein. As used herein, a "target sequence" refers to a contiguous portion of a nucleotide sequence of an mRNA molecule formed during transcription of the ALAS1 gene, including an mRNA that is a product of RNA processing of a primary transcript. The target portion of the sequence will be long enough to be a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be 9-36 nucleotides in length, such as 15-30 nucleotides in length, including all subranges therebetween. As non-limiting examples, the target sequence may be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, The amino acid sequence may be an nucleotide sequence, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0101] As used herein, a "nucleic acid therapeutic agent" is understood to be a therapeutic agent that contains nucleotides of sufficient length to specifically hybridize to a target sequence of a target nucleic acid in a cell, and that hybridization reduces the level of the protein encoded by the target nucleic acid, for example, by inhibiting transcription or promoting sequence-specific degradation of the target nucleic acid. Exemplary nucleic acid therapeutic agents include RNAi agents and antisense oligonucleotide agents.

[0102] The terms "antisense polynucleotide drug," "antisense oligonucleotide," "antisense compound," and "antisense drug," as used interchangeably herein, refer to drugs that include single-stranded oligonucleotides that specifically bind to a target nucleic acid molecule via hydrogen bonds (Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) and inhibit expression of the target nucleic acid by an antisense mechanism of action, e.g., RNase H. In some embodiments, the antisense drug is a nucleic acid therapeutic that acts by reducing expression of a target gene, thereby reducing expression of a polypeptide encoded by the target gene. Exemplary antisense drugs that reduce or inhibit expression of ALAS1 include antisense strands that include or consist of the antisense sequence (or the corresponding unmodified antisense sequence) of AD-60489, AD-60519, AD-61193, or AD-60819 (see U.S. Pat. No. 10,119,143, which is incorporated herein by reference in its entirety).

[0103] One exemplary RNAi agent that reduces expression of ALAS1, givosiran, is provided, for example, in U.S. Pat. No. 10,119,143, filed April 4, 2016, entitled "Compositions and methods for inhibiting expression of the ALAS1 gene," the entirety of which is incorporated by reference herein for all purposes.

[0104] [Table 1]

[0105] A, C, G, and U are adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, and uridine-3'-phosphate, respectively; a, c, g, and u are 2'-O-methyl adenosine-3'-phosphate, 2'-O-methyl cytidine-3'-phosphate, 2'-O-methyl guanosine-3'-phosphate, and 2'-O-methyl uridine-3'-phosphate, respectively; Af, Cf, Gf, and U f is 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; dT is 2'-deoxythymidine-3'-phosphate; s is a phosphorothioate linkage, and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl]-4-hydroxyprolinol.

[0106] As used herein, a "subject diagnosed with AHP" is a subject who has been determined by a medical professional to meet clinically determinative levels of ALA and / or PBG and / or a mutation associated with AHP.

[0107] As used herein, "treatment", "treating" and the like are understood as the administration of a therapeutic agent to reduce the rate of progression of a disease or condition or to alleviate at least one sign or symptom in a subject suffering from a disease. In certain embodiments, an indication of disease can be a change in a biomarker from a healthy reference level prior to the onset of overt symptoms of disease. Natural history studies and clinical trials of AHP have demonstrated disease progression if not treated.

[0108] As used herein, "detection of a protein", "detection of a biomarker" etc. is understood as the detection of a protein or a sufficiently large fragment of said protein to determine the identity of the protein, for example by immunological methods, chromatographic methods etc., used. In certain embodiments, detection of a protein may include detection of one or more isoforms of a protein present in a subject, where no distinction is made between various isoforms. In certain embodiments, the detection method is a clinically accepted or validated method.

[0109] Biomarkers for the diagnosis and management of AHP Plasma proteomics and the identification of less invasive biomarkers are emerging as an integral part of modern drug discovery and clinical development. In an attempt to influence this approach, we examined the plasma proteome of AHP patients in a clinical proteomic study of AHP. This proteomic approach demonstrated that 212 plasma proteins out of 1196 proteins investigated were significantly different between healthy controls and AHP patients. Among these 212 plasma proteins, the three proteins with the largest effect size difference between AHP patients and healthy controls include APLP1, KIM1, and MMP7 (Figure 1A-1B). Two of the three proteins with the largest effect size between AHP patients and healthy controls (KIM1, MMP7) are biomarkers of kidney injury. Based on the measured plasma proteins and previous plasma levels and characterization of kidney injury, we explored additional kidney injury biomarkers. Three additional kidney injury biomarkers (NGAL, CST3, CHI3L1) showed significant elevations in AHP patients.

[0110] The demonstration that plasma levels of renal injury biomarkers are significantly increased in AHP patients is relevant for the diagnosis, treatment, and monitoring of AHP progression. This result is particularly compelling since these biomarkers are elevated in AHP patients with recurrent acute attacks as well as in chronic high excretors (CHE) (Figures 2A, 3A, 4A, 5A, and 6A).

[0111] Correlations were found between the levels of renal injury biomarkers and eGFR in patients with AHP (Figures 2B, 3B, 4B, 5B, and 6B). Glomerular filtration rate (GFR) is a measure of renal function. Testing the actual GFR can be a complex and lengthy procedure, so an estimate of GFR (eGFR) is usually calculated. Accurate estimation of GFR is important for early identification of renal disease, which often has no symptoms until the kidneys fail. Correlations between eGFR and renal injury biomarker levels in patients with AHP are associated with the severity of AHP and disease progression. Correlations were also found between the levels of renal injury biomarkers and AHP patients who had a history or had been previously diagnosed with renal failure and dysfunction.

[0112] As demonstrated herein, the levels of renal injury biomarkers are elevated in AHP patients. In addition to monitoring disease regression upon treatment, the levels of renal injury proteins may serve as potential biomarkers for other aspects of AHP disease. If the levels of renal injury biomarkers are elevated during the early pathogenesis of AHP, they may serve as prognostic indicators for the development of symptomatic disease, such as acute attacks, compared to CHE. In addition, the correlation of the levels of renal injury biomarkers with eGFP suggests that the levels of renal injury biomarkers may correlate with the severity of AHP disease. Finally, renal injury biomarkers may potentially play an important role in determining the efficacy of various treatments. This study was based on AHP patients with recurrent acute attacks as well as CHE. Thus, the presence of elevated renal injury markers is not exclusively limited to patients with acute attack porphyria.

[0113] In certain embodiments, the biomarker is selected from one or more (e.g., two, three, four, five, or all) of KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1. In some embodiments, the biomarker comprises KIM1. In some embodiments, the biomarker comprises APLP1. In some embodiments, the biomarker comprises MMP7. In some embodiments, the biomarker comprises NGAL. In some embodiments, the biomarker comprises CST3. In some embodiments, the biomarker comprises CHI3L1. In some embodiments, the biomarker is a renal injury biomarker selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0114] In some embodiments, the levels of a plurality of biomarkers are determined. In some embodiments, the biomarkers include KIM1 and APLP1. In some embodiments, the biomarkers include KIM1 and MMP7. In some embodiments, the biomarkers include KIM1 and NGAL. In some embodiments, the biomarkers include KIM1 and CST3. In some embodiments, the biomarkers include KIM1 and CHI3L1. In some embodiments, the biomarkers include APLP1 and MMP7. In some embodiments, the biomarkers include APLP1 and NGAL. In some embodiments, the biomarkers include APLP1 and CST3. In some embodiments, the biomarkers include APLP1 and CHI3L1. In some embodiments, the biomarkers include MMP7 and NGAL. In some embodiments, the biomarkers include MMP7 and CST3. In some embodiments, the biomarkers include MMP7 and CHI3L1. In some embodiments, the biomarkers include NGAL and CST3. In some embodiments, the biomarkers include NGAL and CHI3L1. In some embodiments, the biomarkers include CST3 and CHI3L1.

[0115] In some embodiments, the biomarkers include KIM1, MMP7, and APLP1. In some embodiments, the biomarkers include KIM1, MMP7, and NGAL. In some embodiments, the biomarkers include KIM1, MMP7, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, and CHI3L1. In some embodiments, the biomarkers include KIM1, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, and NGAL. In some embodiments, the biomarkers include APLP1, MMP7, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers include APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers include MMP7, NGAL, and CST3. In some embodiments, the biomarkers include MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include NGAL, CST3, and CHI3L1.

[0116] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and NGAL. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, CST3, and CHI3L1.

[0117] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP ...

[0118] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, CST3, and CHI3L1.

[0119] In some embodiments, the biomarkers further include KIM1. In some embodiments, the biomarkers further include APLP1. In some embodiments, the biomarkers further include MMP7. In some embodiments, the biomarkers further include NGAL. In some embodiments, the biomarkers further comprise CST3. In some embodiments, the biomarkers further include CHI3L1.

[0120] Biomarkers for managing the progression of AHP Genetic testing is available to identify subjects with AHP-associated mutations. The present disclosure provides biomarkers, such as kidney damage biomarkers, to determine when subjects with a predisposition to AHP, such as subjects with a genetic predisposition or subjects with elevated ALA and / or PBG, may be treated with drugs that reduce the expression of ALAS1.

[0121] In certain embodiments, the biomarker is selected from one or more (e.g., two, three, four, five, or all) of KIM1, APLP1, MMP7, NGAL, CST3, or CHI3L1. In some embodiments, the biomarker comprises KIM1. In some embodiments, the biomarker comprises APLP1. In some embodiments, the biomarker comprises MMP7. In some embodiments, the biomarker comprises NGAL. In some embodiments, the biomarker comprises CST3. In some embodiments, the biomarker comprises CHI3L1. In some embodiments, the biomarker is a renal injury biomarker selected from one or more (e.g., two, three, four, or all) of KIM1, MMP7, NGAL, CST3, or CHI3L1.

[0122] In some embodiments, the levels of a plurality of biomarkers are determined. In some embodiments, the biomarkers include KIM1 and APLP1. In some embodiments, the biomarkers include KIM1 and MMP7. In some embodiments, the biomarkers include KIM1 and NGAL. In some embodiments, the biomarkers include KIM1 and CST3. In some embodiments, the biomarkers include KIM1 and CHI3L1. In some embodiments, the biomarkers include APLP1 and MMP7. In some embodiments, the biomarkers include APLP1 and NGAL. In some embodiments, the biomarkers include APLP1 and CST3. In some embodiments, the biomarkers include APLP1 and CHI3L1. In some embodiments, the biomarkers include MMP7 and NGAL. In some embodiments, the biomarkers include MMP7 and CST3. In some embodiments, the biomarkers include MMP7 and CHI3L1. In some embodiments, the biomarkers include NGAL and CST3. In some embodiments, the biomarkers include NGAL and CHI3L1. In some embodiments, the biomarkers include CST3 and CHI3L1.

[0123] In some embodiments, the biomarkers include KIM1, MMP7, and APLP1. In some embodiments, the biomarkers include KIM1, MMP7, and NGAL. In some embodiments, the biomarkers include KIM1, MMP7, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, and CHI3L1. In some embodiments, the biomarkers include KIM1, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, and NGAL. In some embodiments, the biomarkers include APLP1, MMP7, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers include APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers include MMP7, NGAL, and CST3. In some embodiments, the biomarkers include MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include NGAL, CST3, and CHI3L1.

[0124] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and NGAL. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP1, NGAL, CST3, and CHI3L1.

[0125] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include KIM1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers include APLP ...

[0126] In some embodiments, the biomarkers include KIM1, APLP1, MMP7, NGAL, CST3, and CHI3L1.

[0127] In some embodiments, the biomarkers further include KIM1. In some embodiments, the biomarkers further include APLP1. In some embodiments, the biomarkers further include MMP7. In some embodiments, the biomarkers further include NGAL. In some embodiments, the biomarkers further comprise CST3. In some embodiments, the biomarkers further include CHI3L1.

[0128] In certain embodiments, after a subject is identified as having an AHP-associated mutation, the subject is routinely monitored for an increase in the level of the biomarker (e.g., a renal biomarker) compared to a reference level, either a population control or the level of the biomarker (e.g., a renal biomarker) in the same subject. An increase in the biomarker (e.g., a renal biomarker) in the subject is an indication that treatment of the subject should be initiated, for example, treatment with a drug that reduces the expression of ALAS1. In certain embodiments, the subject is also routinely monitored for the development of signs or symptoms of AHP. In certain embodiments, the subject is also monitored for the level of one or more of ALA and PBG, where an increase in the level of the biomarker (e.g., a renal biomarker) compared to the reference level indicates worsening AHP if the beta coefficient is positive, and a decrease in the level of the biomarker (e.g., a renal biomarker) compared to the reference level indicates worsening AHP if the beta coefficient is negative.

[0129] Monitoring a biomarker (e.g., a renal biomarker) can also be used to determine whether AHP is progressing in a subject, where an increase in the level of the biomarker (e.g., a renal biomarker) in the subject indicates progression of AHP. Progression can also be monitored by further determining the level of one or more of ALA and PBG, where an increase in the level of the biomarker (e.g., a renal biomarker) compared to a reference level indicates worsening AHP when the beta coefficient is positive, and a decrease in the level of the biomarker (e.g., a renal biomarker) compared to a reference level indicates worsening AHP when the beta coefficient is negative.

[0130] Establishing reference standards for biomarker levels The present disclosure provides for measuring the level of one or more biomarkers (e.g., protein biomarkers) in a subject and comparing the level of the biomarker to its corresponding reference level to determine whether there is a difference between the level of the biomarker and its corresponding reference level. It is understood that the method of determining the level of the biomarker in the sample and the method of determining the reference level for the reference level should be the same. It is further understood that the change from the reference level may be a change in a defined concentration of the biomarker in the sample, e.g., pg / ml of the sample. Alternatively, the change may be a relative amount, such as a percentage change from the reference sample, e.g., at least 150% or at least 200% of the reference sample. The change in level should be statistically significant. The method of determining the level of the biomarker is typically performed in vitro and is often performed in a clinical laboratory when used for diagnostic methods and when used for methods used to select a treatment for a subject.

[0131] Commercially available kits are available for determining the levels of some biomarkers, such as NT-proBNP and troponin I. The levels of these markers can be performed in clinical laboratories using commercially available diagnostic tests, such as chemiluminescence assays (for NT-proBNP, Roche Diagnostic Cobas, Indianapolis, IN, USA; for troponin I, Siemens Centaur XP, Camberley, Surrey, UK). In such cases, reference levels of the biomarkers, and in embodiments, appropriate controls, can be provided by the kit manufacturer.

[0132] Past studies have demonstrated that, in general, there is no single reference level that is appropriate for all subjects. Instead, appropriate age and sex matched control reference standards can be selected for comparison to population-based controls. Such considerations are understood in the art.

[0133] When the subject's previous time point is used as the reference level, it is necessary to provide a sufficient interval to allow the change in the level of the biomarker.Changes in the level of the renal impairment biomarker are observed in both the control subject, whose renal impairment biomarker level is elevated, and the treated subject, whose level is reduced, on day 0 and at one or more time points thereafter.A sufficient interval for the change in the level of the renal impairment biomarker may be determined for each renal impairment biomarker.Depending on the specific biomarker, disease state, and progression rate in the subject, it is expected that a short interval, such as, for example, 6 months, 3 months, 2 months, 1 month, or less than 1 month, may be sufficient to observe the change in the level of the renal impairment marker in the subject.

[0134] Signs and Symptoms of Porphyria The method disclosed herein may further comprise monitoring the subject for one or more signs or symptoms indicative of AHP or the progression of AHP, including but not limited to abdominal pain, pain in the extremities, back pain, chest pain, nausea, vomiting, confusion, anxiety, seizures, constipation, diarrhea, dark red urine, or any combination thereof. The comorbidities indicative of AHP or the progression of AHP that may be monitored include, for example, hypertension, chronic kidney disease, and combinations thereof. The onset or progression of one or more signs or symptoms is further diagnostic of AHP in the context of an elevated level of renal damage marker compared to a reference level.

[0135] Nucleic acid therapeutics that reduce ALAS1 expression In some embodiments, the methods described herein involve the use of nucleic acid therapeutics (e.g., RNAi agents) that reduce expression of ALAS1. In certain embodiments, expression of the ALAS1 gene is reduced or inhibited using an ALAS1-specific iRNA.

[0136] Described herein are compositions and methods that result in RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the ALAS1 gene, such as in a cell or a subject (eg, a mammal, such as a human subject).

[0137] The iRNAs included in the compositions featured herein include, for example, dsRNAs having an RNA strand (antisense strand) that has a region of 30 nucleotides or less in length, typically 19-24 nucleotides, that is substantially complementary to at least a portion of an mRNA transcript of an ALAS1 gene (e.g., mouse or human ALAS1 gene) (also referred to herein as "ALAS1-specific iRNAs"). Alternatively, or in combination, the iRNAs include dsRNAs having an RNA strand (antisense strand) that has a region of 30 nucleotides or less in length, typically 19-24 nucleotides, that is substantially complementary to at least a portion of an mRNA transcript of an ALAS1 gene (e.g., human variant 1 or 2 of the ALAS1 gene) (also referred to herein as "ALAS1-specific iRNAs").

[0138] In an embodiment, an iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region substantially complementary to a region of human ALAS1. In an embodiment, human ALAS1 has the sequence of NM_000688.4 (SEQ ID NO: 11). In an embodiment, human ALAS1 has the sequence of NM_199166.1 (SEQ ID NO: 12). In an embodiment, the antisense sequence of an iRNA (e.g., dsRNA) targets within a region of 871 to 895 (5, 4, 3, 2, or 1 nucleotide, plus or minus, either or both toward the 5' end and / or the 3' end) of the ALAS1 transcript NM_000688.4. In an embodiment, the antisense sequence targets nucleotides 871 to 893, 871 to 892, or 873 to 895 of the ALAS1 transcript NM_000688.4. In embodiments, the antisense sequence comprises or consists of a sequence that is fully complementary or substantially complementary to nucleotides 871 to 893, 871 to 892, or 873 to 895 of the ALAS1 transcript NM_000688.4.

[0139] In one embodiment, a double-stranded ribonucleic acid (dsRNA) that inhibits expression of ALAS1 is provided, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an RNA transcript of ALAS1, and the antisense strand comprises at least 15 (e.g., at least 16, 17, 18, 19, 221, 22, or 23) consecutive nucleotides that differ from the sequence UAAGAUGAGACACUCUUUCUGGU (SEQ ID NO: 13) or UAAGAUGAGACACUCTUUCUGGU (SEQ ID NO: 14) by no more than 3, no more than 2, or no more than 1 nucleotide. In an embodiment, the antisense strand comprises the sequence UAAGAUGAGACACUCUUUCUGGU (SEQ ID NO: 13) or UAAGAUGAGACACUCTUUCUGGU (SEQ ID NO: 14). In an embodiment, the sense strand comprises the sequence CAGAAAGAGUGUCUCAUCUUA (SEQ ID NO: 15). In an embodiment, one or more nucleotides of the antisense strand and / or the sense strand are modified as described herein.

[0140] In some embodiments, the dsRNA is effective to suppress levels of ALAS1 mRNA in the liver, e.g., to achieve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% silencing (e.g., such that the level of ALAS1 mRNA is reduced to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the control level of ALAS1 mRNA in the liver, e.g., the level in an untreated individual or group of individuals, e.g., an individual or group of individuals treated with PBS only).

[0141] In some embodiments, the dsRNA is effective to suppress levels of circulating ALAS1 mRNA, e.g., to achieve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% silencing (e.g., such that the level of ALAS1 mRNA is reduced to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of a control level of circulating ALAS1 mRNA, e.g., the level before dsRNA treatment, or the level in an untreated individual or group of individuals).

[0142] The iRNA molecules featured herein may comprise naturally occurring nucleotides or may comprise at least one modified nucleotide. In embodiments, the at least one modified nucleotide comprises one or more nucleotide modifications selected from the group consisting of locked nucleic acid (LNA), acyclic nucleotide, hexitol or hexose nucleic acid (HNA), cyclohexene nucleic acid (CeNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, or any combination thereof. In certain embodiments, the at least one modified nucleotide comprises, but is not limited to, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, nucleotide with 5'-phosphorothioate group, and terminal nucleotide linked to a ligand such as, for example, N-acetylgalactosamine (GalNAc) or cholesteryl derivative.

[0143] Alternatively, the modified nucleotide may be selected from the group of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, acyclic nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0144] In some embodiments, the region of complementarity is at least 17 nucleotides in length. In some embodiments, the region of complementarity is between 19 and 21 nucleotides in length. In some embodiments, the region of complementarity is 19 nucleotides in length. In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, at least one strand comprises a 3' overhang of at least one nucleotide. In embodiments, the antisense strand comprises a 3' overhang of at least one nucleotide.

[0145] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides. In embodiments, the antisense strand comprises a 3' overhang of at least 2 nucleotides. In embodiments, the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0146] In certain embodiments, the iRNAs described herein target a wild-type ALAS1 RNA transcript variant, and in other embodiments, the iRNA targets a mutant transcript (e.g., an ALAS1 RNA having an allelic variant). For example, the iRNAs featured in this disclosure may target a polymorphic variant, such as a single nucleotide polymorphism (SNP), of ALAS1. In other embodiments, the iRNA targets both wild-type and mutant ALAS1 transcripts. In yet other embodiments, the iRNA targets a specific transcript variant of ALAS1 (e.g., human ALAS1 variant 1). In yet other embodiments, the iRNA agent targets multiple transcript variants (e.g., both variant 1 and variant 2 of human ALAS1).

[0147] In some embodiments, the iRNAs featured in this disclosure target non-coding regions of ALAS1 RNA transcripts, such as the 5' or 3' untranslated regions of the transcripts. In some embodiments, the iRNAs described herein are in the form of conjugates, which may act as targeting moieties and / or ligands, as described herein, such as carbohydrate conjugates. In some embodiments, the conjugates are attached to the 3' end of the sense strand of the dsRNA. In some embodiments, the conjugates are attached via linkers, such as bivalent or trivalent branched linkers.

[0148] In some embodiments, the conjugate comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Such conjugates are also referred to herein as GalNAc conjugates. In some embodiments, the conjugate targets the RNAi agent to a specific cell, such as, for example, liver cells, e.g., hepatocytes. The GalNAc derivative can be conjugated via a linker, such as, for example, a bivalent or trivalent branched linker. In some embodiments, the RNAi agent is conjugated to the carbohydrate conjugate via a linker.

[0149] In some embodiments, the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an ALAS1 RNA transcript, the sense strand comprises the sequence of csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 16), and all of its modifications, and the antisense strand comprises the sequence of usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsgsu (SEQ ID NO: 17), and all of its modifications; where c, a, g, u = 2'-OMe ribonucleosides; Af, Cf, Gf, Uf = 2'F ribonucleosides; s = phosphorothioates, and where [ka]

[0150] In some embodiments, the dsRNA comprises a duplex region 21-23 nucleotide pairs in length. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides. In some embodiments, each strand is 26 nucleotides or less in length.

[0151] In some embodiments, the antisense strand consists of the sequence: usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsgsu (SEQ ID NO: 17). In some embodiments, the sense strand consists of the sequence: csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 16). In some embodiments, the sense strand consists of the sequence: csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 16) and the antisense strand consists of the sequence: usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsgsu (SEQ ID NO: 17).

[0152] In some embodiments, the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to an ALAS1 RNA transcript, and the dsRNA is in the form of a conjugate having the following structure: [ka] or a pharma- ceutically acceptable salt thereof; where Af, Cf, Gf, Uf = 2'F ribonucleosides; Am, Cm, Gm, Um = 2'-OMe ribonucleosides; [ka] [ka] and [ka]

[0153] In some embodiments, the dsRNA comprises a duplex region 21 nucleotide pairs in length. In some embodiments, the antisense strand comprises a 2-nucleotide 3' overhang.

[0154] Other exemplary nucleic acid therapeutics that reduce the expression of ALAS1 are described in International Publication Nos. WO2013 / 155204 and WO2015 / 051318, U.S. Patent Nos. 9,133,461, 9,631,193, 10,400,239, 10,119,143, 10,125,364, and 11,028,392, and U.S. Application Publication No. US2013 / 0281511. Nos. 2015 / 0111841, 2016 / 0115476, 2016 / 0244766, 2018 / 0037886, 2019 / 0144870, 2019 / 0218549, 2020 / 0181614 and 2021 / 0087558, the entire contents of each of which are incorporated by reference.

[0155] An aspect provided herein is a composition, e.g., a pharmaceutical composition, comprising one or more of the iRNAs described herein and a pharma- ceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used to inhibit the expression of the ALAS1 gene in an organism, typically a human subject. In one embodiment, the composition is used to treat porphyria, e.g., AHP, e.g., AIP.

[0156] In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is administered in an unbuffered solution. In embodiments, the unbuffered solution is saline or water, e.g., water for injection.

[0157] In some embodiments, the pharmaceutical composition comprises an iRNA and water for injection. In embodiments, the composition comprises about 100 to 300 mg / ml, e.g., 200 mg / ml, of an iRNA. In embodiments, the composition has a pH of 6.0-7.5, e.g., about 7.0. In embodiments, the composition is for subcutaneous injection. In embodiments, the pharmaceutical composition is packaged in a container (e.g., a glass vial, e.g., a 2 mL glass vial) in a volume of about 0.3 to 1 mL, e.g., 0.55 mL.

[0158] In an embodiment of the pharmaceutical composition described herein, the iRNA (e.g., dsRNA) is administered in a buffer solution. In an embodiment, the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In an embodiment, the buffer solution is phosphate buffered saline (PBS).

[0159] In embodiments of the pharmaceutical compositions described herein, the iRNA (eg, dsRNA) is targeted to liver cells.

[0160] In embodiments of the pharmaceutical compositions described herein, the compositions are administered intravenously. In embodiments of the pharmaceutical compositions described herein, the compositions are administered subcutaneously.

[0161] In some embodiments, dsRNA is administered at a dosage of 0.01mg / kg to 5mg / kg, or 1mg / kg to 2.5mg / kg (subject's body weight) monthly.In some embodiments, dsRNA is administered at a dosage of 2.5mg / kg (subject's body weight) monthly.

[0162] In embodiments, the pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that includes a ligand (e.g., a GalNAc ligand) that targets the iRNA (e.g., a dsRNA) to hepatocytes.

[0163] In an embodiment, the pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that includes a ligand (e.g., a GalNAc ligand), and the pharmaceutical composition is administered subcutaneously. In an embodiment, the ligand targets the iRNA (e.g., a dsRNA) to hepatocytes.

[0164] In certain embodiments, pharmaceutical compositions, such as compositions described herein, comprise lipid formulations.In some embodiments, RNAi agents are included in LNP formulations, such as MC3 formulations.In some embodiments, LNP formulations target RNAi agents to specific cells, such as liver cells, such as hepatocytes.In some embodiments, lipid formulations are LNP11 formulations.In some embodiments, the composition is administered intravenously.

[0165] In another embodiment, the pharmaceutical composition is formulated for administration according to a dosing regimen described herein, e.g., not more than once every 4 weeks, not more than once every 3 weeks, not more than once every 2 weeks, or not more than once every week, etc. In another embodiment, administration of the pharmaceutical composition can continue for one month or more, e.g., one month, two months, three months, or six months, or one year or more.

[0166] In another embodiment, a composition containing an iRNA featured in this disclosure, such as a dsRNA targeting ALAS1, is administered in conjunction with a non-iRNA therapeutic agent, such as a drug known to treat porphyria (e.g., AIP) or a symptom of porphyria (e.g., pain). In another embodiment, a composition containing an iRNA featured in this disclosure, such as a dsRNA targeting AIP, is administered in conjunction with a non-iRNA therapeutic regimen, such as hemin or glucose (e.g., glucose infusion (e.g., IV glucose)). For example, an iRNA featured in this disclosure can be administered before, after, or simultaneously with glucose, dextrose, or similar treatments that restore energy balance (e.g., total parenteral nutrition). An iRNA featured in this disclosure can also be administered before, after, or simultaneously with the administration of a heme formulation (e.g., hemin, heme alginate, or albumin-heme), and optionally in combination with glucose (e.g., IV glucose), or the like.

[0167] Typically, glucose administered for the treatment of porphyria is administered intravenously (IV). Intravenous administration of glucose is referred to herein as "IV glucose." However, alternative embodiments in which glucose is administered by other means are also encompassed.

[0168] In one embodiment, an ALAS1 iRNA is administered to a patient and then a non-iRNA drug or therapeutic regimen (e.g., glucose and / or heme formulations) is administered to the patient (or vice versa). In another embodiment, the ALAS1 iRNA and the non-iRNA therapeutic drug or therapeutic regimen are administered simultaneously.

[0169] Further embodiments Disclosed herein is a method for treating a human subject having or at risk of having AHP. The subject may have recurrent acute attacks of AHP. The subject may be a chronic high excretor.

[0170] In some embodiments, the subject has not been diagnosed with porphyria. In some embodiments, the subject has not been diagnosed with AHP. For example, the subject may not have been determined by a medical professional to meet clinically determinative ALA and / or PBG levels and / or mutations associated with AHP.

[0171] In some embodiments, the subject does not meet the diagnostic criteria for AHP. For example, the subject may not meet one or both of the following: (i) having clinically determinative levels of ALA and / or PBG, and (ii) having a mutation associated with AHP.

[0172] In some embodiments, the subject has elevated ALA and / or PBG levels.Elevated ALA levels can be, for example, greater than 35 μmol / L in biological samples, or greater than 60 μmol / d in a 24-hour period.Elevated PBG levels can be, for example, greater than 8.8 μmol / d in a 24-hour period.

[0173] In some embodiments, the subject has a mutation associated with AHP. For example, the subject may have a specific pathogenic gene mutation for AHP (e.g., a mutation in HMBS).

[0174] In some embodiments, the subject does not have a mutation associated with AHP. For example, the subject may not have a specific pathogenic gene mutation for AHP (e.g., a mutation in HMBS).

[0175] In some embodiments, the subject has a history of renal dysfunction. For example, the subject may have been previously diagnosed with renal dysfunction. The subject may have recovered from renal dysfunction. The subject may suffer from renal dysfunction.

[0176] In some embodiments, the subject has been diagnosed with renal impairment. For example, the subject may have been diagnosed with chronic kidney disease or acute kidney injury.

[0177] In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). The subject may have an eGFR at stage 1, stage 2, stage 3, stage 4, or stage 5. Stage 1 (eGFR of 90 or greater) indicates mild kidney damage, but the kidneys are working well. Stage 2 (eGFR between 60 and 89) indicates more advanced kidney damage than stage 1, but the kidneys continue to function well. Stage 3 (eGFR between 30 and 59) indicates a decline in kidney function, and the subject may be experiencing symptoms. Stage 4 (eGFR between 15 and 29) indicates poor kidney function, with moderate to severe kidney damage. Stage 5 (eGFR below 15) is a sign of kidney failure, with less than 15% kidney function.

[0178] In some embodiments, the level of a biomarker (e.g., a renal impairment biomarker) in the subject is elevated by at least 2-fold, e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold, compared to a reference level of the renal impairment biomarker. The reference level may be the level of a healthy control or a previous level in the same subject.

[0179] The level of a biomarker, eg, a renal injury biomarker, may be determined in a subject's sample selected from blood, plasma, serum, urine, or stool.

[0180] In some embodiments, the subject is being treated with a second therapeutic agent. The second therapeutic agent may be an agent for treating AHP. For example, the second therapeutic agent includes a heme preparation (e.g., hemin, heme arginate, or albumin-heme), glucose (e.g., IV glucose), dextrose, or a combination thereof. The second therapeutic agent may be an agent for treating kidney damage. For example, the second therapeutic agent may be an agent for treating chronic kidney damage or acute kidney damage. The second therapeutic agent may be an agent for treating a symptom of AHP. The second therapeutic agent may be an agent for treating a comorbidity of AHP.

[0181] The method may further include discontinuing treatment with the second therapeutic agent if the subject has an elevated level of a biomarker, eg, a renal injury biomarker.

[0182] In some embodiments, the subject further suffers from one or more symptoms associated with AHP. For example, the subject may suffer from abdominal pain, pain in the extremities, back pain, chest pain, nausea, vomiting, confusion, anxiety, seizures, constipation, diarrhea, dark red urine, or any combination thereof.

[0183] In some embodiments, the subject further suffers from one or more comorbidities associated with AHP, for example, the subject may suffer from hypertension, chronic kidney disease, or both. EXAMPLES

[0184] Example 1: Proteins identified as different in AHP patients compared to healthy controls To identify proteomic changes associated with AHP, we performed an observational case-control study in which we compared the proteomes of AHP patients, chronic high excretors (CHE), and healthy controls. The results are shown graphically in Figures 1A-1B.

[0185] In consented AHP patients (>90% AIP) with recurrent acute attacks in the first cohort (n=108) (Natural History Study of AHP, Identifier NCT02240784), in the second cohort (n=85) (Study to Evaluate the Efficacy and Safety of Givosiran in Patients with AHP, Phase 3, Identifier NCT03338816), and in CHE patients (n=22) (Study of Givosiran in AIP, Phase 1, Identifier NCT02452372), 1196 proteins were measured in plasma at baseline using proteomic analysis (OLINK® platform, Olink Proteomics Inc., Watertown, MA). A separate cohort of healthy controls, age- and sex-matched to the patients in cohort 1, was also analyzed. Linear regression taking into account age and sex was used to determine proteins that were significantly different between AHP or CHE patients and healthy controls.

[0186] Plasma levels of proteomic assays were analyzed to discover a set of biomarkers indicative of AHP disease compared to healthy controls, symptomatic disease compared to CHE, and severity of AHP disease.

[0187] Each patient in Cohort 1 and Cohort 2 was compared to a healthy control. A total of 212 plasma proteins were identified that were different in AHP patients compared to healthy controls. The 212 plasma proteins are plotted in the graphs in Figure 1A (Cohort 1) and Figure 1B (Cohort 2).

[0188] As shown in the data presented in Figures 1A-1B, among the 212 plasma proteins, matrix metalloproteinase 7 (MMP7), amyloid-like protein 1 (APLP1), and kidney injury molecule 1 (KIM1) were the proteins with the highest effect sizes between AHP patients and healthy controls. The results were consistent between the two cohorts. Two kidney injury biomarkers, KIM1 and MMP7, were identified as having the highest effect sizes (KIM1; 3.4-fold; p-value=8.0e-13) (MMP7; 5-fold; p-value=1.5e-25).

[0189] Therefore, certain biomarkers, including, for example, KIM1, APLP1, and MMP7, may be useful in the diagnosis and management of kidney disease in patients with AHP.

[0190] Example 2: Biomarkers of renal injury are elevated in AHP patients and CHE compared to healthy controls Biomarkers of kidney damage were found to be significantly different between AHP patients and healthy controls in Example 1. Other biomarkers of kidney damage were further investigated. Results for five of these proteins are shown in the graphs in Figures 2A-2C, 3A-3C, 4A-4C, 5A-5C, and 6A-6C, respectively.

[0191] Plasma protein levels of renal injury biomarkers were measured in AHP patients from cohort 1 and cohort 2, as well as in CHE patients, and compared with healthy controls. Both KIM1 and MP7 are significantly elevated in AHP patients and CHE from both cohorts compared with healthy control samples (p-values ​​<0.01; Figures 2A and 3A, respectively).

[0192] KIM1 levels are more significantly elevated in patients in cohort 2 with a history of renal failure and dysfunction (p-value 1e-5; 2.4X; Figure 2B). KIM1 levels correlate well with eGFR (r=0.47) and serum creatinine (r=0.47) (Figure 2C). MMP7 is significantly elevated in patients with a history of renal failure and dysfunction (p-value 0.00035; Figure 3B) and somewhat correlates with eGFR (r=0.36) (Figure 3C).

[0193] Based on the proteins measured by OLINK® in Example 1 and the previous plasma levels and characterization of renal injury, additional renal injury biomarkers were selected to be explored. The renal injury biomarkers Neutrophil Gelatinase-Associated Lipocalin (NGAL), Cystatin 3 (CST3) and Chitinase 3-Like Protein 1 (CHI3L1) were significantly elevated in AHP patients (Figures 4A, 5A, and 6A, respectively). AHP patients diagnosed with renal disease demonstrated significantly higher levels of each of these biomarkers than those without such a diagnosis (p-values ​​<0.01) (Figures 4B, 5B, and 6B, respectively). Moderate to strong correlations were found between each of these biomarkers and eGFR (correlation coefficients -0.33 to -0.54) (Figures 4C, 5C, and 6C, respectively). CST3 was also significantly elevated in CHE patients compared to controls (Figure 5A).

[0194] As shown in the data presented in Figures 2A-2C, 3A-3C, 4A-4C, 5A-5C, and 6A-6C, the renal injury biomarkers KIM1, MMP7, NGAL, CST3, and CHI3L1 were found to be elevated in AHP patients compared to healthy controls, KIM1, MMP7, and CST3 were also found to be elevated in CHE patients, and KIM1, MMP7, NGAL, CST3, and CHI3L1 were further elevated in patients with a previous diagnosis of renal injury and were moderately correlated with eGFR.

[0195] Therefore, renal injury biomarkers may be useful in diagnosing and managing kidney disease in AHP patients with recurrent acute attacks as well as in chronic high excretors. These biomarkers may be useful in diagnosing and monitoring renal injury in AHP patients.

[0196] Exemplary Sequences KIM1 Locus NP_036338 364 aa Linear PRI 26-SEP-2021 Definition Hepatitis A virus receptor 1 isoform a precursor [Homo sapiens] Accession number NP_036338 Version NP_036338.2 1 mhpqvvilsl ilhladsvag svkvggeagp svtlpchysg avtsmcwnrg scslftcqng 61 ivwtngthvt yrkdtrykll gdlsrrdvsl tientavsds gvyccrvehr gwfndmkitv 121 sleivppkvt ttpivttvpt vttvrtsttv pttttvpmtt vptttvpttm sipttttvlt 181 tmtvstttsv ptttsipttt svpvtttvst fvppmplprq nhepvatsps spqpaethpt 241 tlqgairrep tssplysytt dgndtvtess dglwnnnqtq lflehsllta nttkgiyagv 301 cisvlvllal lgviiakkyf fkkevqqlsv sfsslqikal qnavekevqa edniyiensl 361 yatd (SEQ ID NO:1)

[0197] Locus NP_001166864 364 aa Linear PRI 26-SEP-2021 Definition Hepatitis A virus receptor 1 isoform a precursor [Homo sapiens] Accession number NP_001166864 Version NP_001166864.1 1 mhpqvvilsl ilhladsvag svkvggeagp svtlpchysg avtsmcwnrg scslftcqng 61 ivwtngthvt yrkdtrykll gdlsrrdvsl tientavsds gvyccrvehr gwfndmkitv 121 sleivppkvt ttpivttvpt vttvrtsttv pttttvpmtt vptttvpttm sipttttvlt 181 tmtvstttsv ptttsipttt svpvtttvst fvppmplprq nhepvatsps spqpaethpt 241 tlqgairrep tssplysytt dgndtvtess dglwnnnqtq lflehsllta nttkgiyagv 301 cisvlvllal lgviiakkyf fkkevqqlsv sfsslqikal qnavekevqa edniyiensl 361 yatd (SEQ ID NO:2)

[0198] Locus NP_001295085 364 aa Linear PRI 26-SEP-2021 Definition Hepatitis A virus receptor 1 isoform b precursor [Homo sapiens] Accession numbers NP_001295085 and XP_011532809 Version NP_001295085.1 1 mhpqvvilsl ilhladsvag svkvggeagp svtlpchysg avtsmcwnrg scslftcqng 61 ivwtngthvt yrkdtrykll gdlsrrdvsl tientavsds gvyccrvehr gwfndmkitv 121 sleivppkvt ttpivttvpt vttvrtsttv pttttvpmtt vptttvpttm sipttttvlt 181 tmtvstttsv ptttsipttt svpvtttvst fvppmplprq nhepvatsps spqpaethpt 241 tlqgairrep tssplysytt dgndtvtess dglwnnnqtq lflehsllta nttkgiyagv 301 cisvlvllal lgviiakmfh laafklklck mqlkrkskqk tistlrivfm prtktqwcsl 361 rvyahecrrl nrhqhirrll dpktiflfqf hlafqhvsdt g (SEQ ID NO:3)

[0199] MMP7 Locus NP_002414 267 aa Linear PRI 19-SEP-2021 Definition Matrilysin preproprotein [Homo sapiens] Accession number NP_002414 Version NP_002414.1 1 mrltvlcavc llpgslalpl pqeaggmsel qweqaqdylk rfylydsetk nansleaklk 61 emqkffglpi tgmlnsrvie imqkprcgvp dvaeyslfpn spkwtskvvt yrivsytrdl 121 phitvdrlvs kalnmwgkei plhfrkvvwg tadimigfar gahgdsypfd gpgntlahaf 181 apgtglggda hfdederwtd gsslginfly aathelghsl gmghssdpna vmyptygngd 241 pqnfklsqdd ikgiqklygk rsnsrkk (SEQ ID NO:4)

[0200] NGAL Locus NP_005555 198 aa Linear PRI 19-SEP-2021 Definition Neutrophil gelatinase-associated lipocalin precursor [Homo sapiens] Accession number NP_005555 Version NP_005555.2 1 mplgllwlgl allgalhaqa qdstsdlipa pplskvplqq nfqdnqfqgk wyvvglagna 61 ilredkdpqk myatiyelke dksynvtsvl frkkkcdywi rtfvpgcqpg eftlgniksy 121 pgltsylvrv vstnynqham vffkkvsqnr eyfkitlygr tkeltselke nfirfskslg 181 lpenhivfpv pidqcidg (SEQ ID NO:5)

[0201] CST3 Locus NP_000090 146 aa Linear PRI 12-SEP-2021 Definition Cystatin C precursor [Homo sapiens]. Accession number NP_000090 Version NP_000090.1 1 magplrapll llailavala vspaagsspg kpprlvggpm dasveeegvr raldfavgey 61 nkasndmyhs ralqvvrark qivagvnyfl dvelgrttct ktqpnldncp fhdqphlkrk 121 afcsfqiyav pwqgtmtlsk stcqda (SEQ ID NO:6)

[0202] Locus NP_001275543 146 aa Linear PRI 12-SEP-2021 Definition Cystatin C Precursor [Homo sapiens] Accession numbers NP_001275543 and XP_005260729 Version NP_001275543.1 1 magplrapll llailavala vspaagsspg kpprlvggpm dasveeegvr raldfavgey 61 nkasndmyhs ralqvvrark qivagvnyfl dvelgrttct ktqpnldncp fhdqphlkrk 121 afcsfqiyav pwqgtmtlsk stcqda (SEQ ID NO:7)

[0203] CHI3L1 Locus NP_001267 383 aa Linear PRI 19-SEP-2021 Definition Chitinase 3-like protein 1 precursor [Homo sapiens] Accession number NP_001267 Version NP_001267.2 1 mgvkasqtgf vvlvllqccs ayklvcyyts wsqyregdgs cfpdaldrfl cthiiysfan 61 isndhidtwe wndvtlygml ntlknrnpnl ktllsvggwn fgsqrfskia sntqsrrtfi 121 ksvppflrth gfdgldlawl ypgrrdkqhf ttlikemkae fikeaqpgkk qlllsaalsa 181 gkvtidssyd iakisqhldf isimtydfhg awrgttghhs plfrgqedas pdrfsntdya 241 vgymlrlgap asklvmgipt fgrsftlass etgvgapisg pgipgrftke agtlayyeic 301 dflrgatvhr ilgqqvpyat kgnqwvgydd qesvkskvqy lkdrqlagam vwaldlddfq 361 gsfcgqdlrf pltnaikdal aat (SEQ ID NO:8)

[0204] APLP1 Locus NP_005157 650 aa Linear PRI 02-JUL-2021 Definition Amyloid-like protein 1 isoform 2 precursor [Homo sapiens] Accession number NP_005157 Version NP_005157.1 1 mgpaspaarg lsrrrpgqppl plllplllll lraqpaigsl aggspgaaea pgsaqvaglc 61 grltlhrdlr tgrwepdpqr srrclrdpqr vleycrqmyp elqiarveqa tqaipmerwc 121 ggsrsgscah phhqvvpfrc lpgefvseal lvpegcrflh qermdqcess trrhqeaqea 181 cssqglilhg sgmllpcgsd rfrgveyvcc pppgtpdpsg tavgdpstrs wppgsrvega 241 edeeeeesfp qpvddyfvep pqaeeeeetv pppsshtlav vgkvtptprp tdgvdiyfgm 301 pgeisehegf lrakmdleer rmrqinevmr ewamadnqsk nlpkadrqal nehfqsilqt 361 leeqvsgerq rlvethatrv ialindqrra alegflaalq adppqaervl lalrrylrae 421 qkeqrhtlrh yqhvaavdpe kaqqmrfqvh thlqvieerv nqslglldqn phlaqelrpq 481 iqellhsehl gpseleapap ggssedkggl qppdskddtp mtlpkgsteq daaspekekm 541 npleqyerkv nasvprgfpf hsseiqrdel apagtgvsre avsgllimga gggslivlsm 601 lllrrkkpyg aishgvvevd pmltleeqql relqrhgyen ptyrfleerp (SEQ ID NO: 9)

[0205] Locus NP_001019978 651 aa Linear PRI 01-JUL-2021 Definition Amyloid-like protein 1 isoform 1 precursor [Homo sapiens] Accession number NP_001019978 Version NP_001019978.1 1 mgpaspaarg lsrrrpgqppl plllplllll lraqpaigsl aggspgaaea pgsaqvaglc 61 grltlhrdlr tgrwepdpqr srrclrdpqr vleycrqmyp elqiarveqa tqaipmerwc 121 ggsrsgscah phhqvvpfrc lpgefvseal lvpegcrflh qermdqcess trrhqeaqea 181 cssqglilhg sgmllpcgsd rfrgveyvcc pppgtpdpsg tavgdpstrs wppgsrvega 241 edeeeeesfp qpvddyfvep pqaeeeeetv pppsshtlav vgkvtptprp tdgvdiyfgm 301 pgeisehegf lrakmdleer rmrqinevmr ewamadnqsk nlpkadrqal nehfqsilqt 361 leeqvsgerq rlvethatrv ialindqrra alegflaalq adppqaervl lalrrylrae 421 qkeqrhtlrh yqhvaavdpe kaqqmrfqvh thlqvieerv nqslglldqn phlaqelrpq 481 iqellhsehl gpseleapap ggssedkggl qppdskdadt pmtlpkgste qdaaspekek 541 mnpleqyerk vnasvprgfp fhsseiqrde lapagtgvsr eavsgllimg agggslivls 601 mlllrrkkpy gaishgvvev dpmltleeqq lrelqrhgye nptyrfleer p (SEQ ID NO: 10)

[0206] ALAS1 Locus NM_000688 2407 bp mRNA linear PRI 19-NOV-2011 Definition: Homo sapiens aminolevulinic acid, delta-, synthase 1 (ALAS1), Transcript variant 1, mRNA Accession NM_000688 Version NM_000688.4 1 ctgtatatta aggcgccggc gatcgcggcc tgaggctgct cccggacaag ggcaacgagc 61 gtttcgtttg gacttctcga cttgagtgcc cgctctcttc gccgccgcct ctgcagtcct 121 cagcgcagtt atgcccagtt cttcccgctg tggggacacg accacggagg aatccttgct 181 tcaggactc gggaccctgc tggacccctt cctcgggttt aggggatgtg gggaccagga 241 gaaagtcagg atccctaaga gtcttccctg cctggatgga tgagtggctt cttctccacc 301 tagattcttt ccacaggagc cagcatactt cctgaacatg gagagtgttg ttcgccgctg 361 cccattctta tcccgagtcc cccaggcctt tctgcagaaa gcaggcaaat ctctgttgtt 421 ctatgcccaa aactgcccca agatgatgga agttggggcc aagccagccc ctcgggcatt 481 gtccactgca gcagtacact accaacagat caaagaaacc cctccggcca gtgagaaaga 541 caaaactgct aaggccaagg tccaacagac tcctgatgga tcccagcaga gtccagatgg 601 cacacagctt ccgtctggac accccttgcc tgccacaagc cagggcactg caagcaaatg 661 ccctttcctg gcagcacaga tgaatcagag aggcagcagt gtcttctgca aagccagtct 721 tgagcttcag gaggatgtgc aggaaatgaa tgccgtgagg aaagaggttg ctgaaacctc 781 agcaggcccc agtgtggtta gtgtgaaaac cgatggaggg gatcccagtg gactgctgaa 841 gaacttccag gacatcatgc aaaagcaaag accagaaaga gtgtctcatc ttcttcaaga 901 taacttgcca aaatctgttt ccacttttca gtatgatcgt ttctttgaga aaaaaattga 961 tgagaaaaag aatgaccaca cctatcgagt ttttaaaact gtgaaccggc gagcacacat 1021 cttccccatg gcagatgact attcagactc cctcatcacc aaaaagcaag tgtcagtctg 1081 gtgcagtaat gactacctag gaatgagtcg ccacccacgg gtgtgtgggg cagttatgga 1141 cactttgaaa caacatggtg ctggggcagg tggtactaga aatatttctg gaactagtaa 1201 attccatgtg gacttagagc gggagctggc agacctccat gggaaagatg ccgcactctt 1261 gttttcctcg tgctttgtgg ccaatgactc aaccctcttc accctggcta agatgatgcc 1321 aggctgtgag atttactctg attctgggaa ccatgcctcc atgatccaag ggattcgaaa 1381 cagccgagtg ccaaagtaca tcttccgcca caatgatgtc agccacctca gagaactgct 1441 gcaaagatct gacccctcag tccccaagat tgtggcattt gaaactgtcc attcaatgga 1501 tggggcggtg tgcccactgg aagagctgtg tgatgtggcc catgagtttg gagcaatcac 1561 cttcgtggat gaggtccacg cagtggggct ttatggggct cgaggcggag ggattggggga 1621 tcgggatgga gtcatgccaa aaatggacat catttctgga acacttggca aagcctttgg 1681 ttgtgttgga gggtacatcg ccagcacgag ttctctgatt gacaccgtac ggtcctatgc 1741 tgctggcttc atcttcacca cctctctgcc acccatgctg ctggctggag ccctggagtc 1801 tgtgcggatc ctgaagagcg ctgagggacg ggtgcttgc cgccagcacc agcgcaacgt 1861 caaactcatg agacagatgc taatggatgc cggcctccct gttgtccact gccccagcca 1921 catcatccct gtgcgggtttg cagatgctgc taaaaacaca gaagtctgtg atgaactaat 1981 gagcagacat aacatctacg tgcaagcaat caattaccct acggtgcccc ggggaaaga 2041 gctcctacgg attgccccca cccctcacca cacaccccag atgatgaact acttccttga 2101 gaatctgcta gtcacatgga agcaagtggg gctggaactg aagcctcatt cctcagctga 2161 gtgcaacttc tgcaggaggc cactgcattt tgaagtgatg agtgaaagag agaagtccta 2221 tttctcaggc ttgagcaagt tggtatctgc tcaggcctga gcatgacctc aattatttca 2281 cttaacccca ggccattatc atatccagat ggtcttcaga gttgtcttta tatgtgaatt 2341 aagttatatt aaattttaat ctatagtaaa aacatagtcc tggaaataaa ttcttgctta 2401 aatggtg (SEQ ID NO:11)

[0207] Locus NM_199166 2258 bp mRNA linear PRI 19-NOV-2011 Definition: Homo sapiens aminolevulinic acid, delta-, synthase 1 (ALAS1), Transcript variant 2, mRNA Accession NM_199166 Version NM_199166.1 1 caaggcgcat gcgcagcggt cactcccgct gtatattaag gcgccggcga tcgcggcctg 61 aggctgctcc cggacaaggg caacgagcgt ttcgtttgga cttctcgact tgagtgcccg 121 cctccttcgc cgccgcctct gcagtcctca gcgcagtctt tccacaggag ccagcatact 181 tcctgaacat ggagagtgtt gttcgccgct gcccattctt atcccgagtc ccccaggcct 241 ttctgcagaa agcaggcaaa tcttgttgt tctatgcca aaactgcccc aagatgatgg 301 aagttggggc caagccagcc cctcgggcat tgtccactgc agcagtacac taccacaga 361 tcaaagaac cccctccggcc agtgagaag acaaactgc taaggccaag gtccaacaga 421 ctcctgatgg atcccagcag agtccagatg gcacacagct tccgtctgga caccccttgc 481 ctgccacag ccaggcact gcaagcaat gcctttcct ggcagcacag atgaatcaga 541 gaggcagcag tgtctctgc aaagccagtc tgagctca ggaggatgtg caggaatga 601 atgccgtgag gaagaggtt gctgaaacct cagcaggccc cagtgtggtt agtgtgaaaa 661 ccgatggagg ggatcccagt ggactgctga agaacttcca ggacatg caaagcaa 721 gaccagaaag agtgtctcat cttcttcaag ataacttgcc aaaatctgtt tccactttc 781 agtatgatcg tttcttgag aaaaaaattg atgagaaaa gatgaccac acctatcgag 841 ttttaaac tgtgaccgg cgagcacaca tctcccat ggcagatgac tattcagact 901 ccctcatcac caaaagcaa gtgtcagtct ggtgcagtaa tgactaccta ggatgagtc 961 gccacccag ggtgtgtggg gcagttatgg acactttgaa acaacatggt gctggggcag 1021 1081 cagacctcca tgggaaagat gccgcactct tgttttcctc gtgctttgtg gccaatgact 1141 caaccctctt caccctggct aagatgatgc caggctgtga gatttactct gattctggga 1201 accatgcctc catgatccaa gggattcgaa acagccgagt gccaaagtac atcttccgcc 1261 acaatgatgt cagccacctc agagaactgc tgcaaagatc tgacccctca gtccccaaga 1321 ttgtggcatt tgaaactgtc cattcaatgg atggggcggt gtgcccactg gaagagctgt 1381 gtgatgtggc ccatgagttt ggagcaatca ccttcgtgga tgaggtccac gcagtggggc 1441 tttatggggc tcgaggcgga gggattgggg atcgggatgg agtcatgcca aaaatggaca 1501 tcatttctgg aacacttggc aaagcctttg gttgtgttgg agggtacatc gccagcacga 1561 gttctctgat tgacaccgta cggtcctatg ctgctggctt catcttcacc acctctctgc 1621 cacccatgct gctggctgga gccctggagt ctgtgcggat cctgaagagc gctgagggac 1681 gggtgcttcg ccgccagcac cagcgcaacg tcaaactcat gagacagatg ctaatggatg 1741 ccggcctccc tgttgtccac tgccccagcc acatcatccc tgtgcgggtt gcagatgctg 1801 ctaaaaacac agaagtctgt gatgaactaa tgagcagaca taacatctac gtgcaagcaa 1861 tcaattaccc tacggtgccc cggggagaag agctcctacg gattgccccc acccctcacc 1921 acacacccca gatgatgaac tacttccttg agaatctgct agtcacatgg aagcaagtgg 1981 ggctggaact gaagcctcat tcctcagctg agtgcaactt ctgcaggagg ccactgcatt 2041 ttgaagtgat gagtgaaaga gagaagtcct atttctcagg cttgagcaag ttggtatctg 2101 ctcaggcctg agcatgacct caattatttc acttaacccc aggccattat catatccaga 2161 tggtcttcag agttgtcttt atatgtgaat taagttatat taaattttaa tctatagtaa 2221 aaacatagtc ctggaaataa attcttgctt aaatggtg (SEQ ID NO: 12)

Claims

1. 1. An in vitro method for determining whether a subject is suffering from acute hepatic porphyria (AHP), the method comprising: (a) determining the level of a biomarker in a sample of a subject; (b) comparing the level of the biomarker determined in step (a) with a reference level of the biomarker; and (c) assessing whether the subject is suffering from AHP, wherein an elevation in the level of the biomarker determined in step (a) compared to a reference level of the biomarker indicates that the subject is suffering from AHP; A method comprising:

2. 2. The method of claim 1, wherein the biomarker is selected from the group consisting of KIM1, APLP1, MMP7, NGAL, CST3, and CHI3L1.

3. 10. The method of claim 1, wherein the biomarker is a renal injury biomarker.

4. The subject has been diagnosed with AHP; The subject has elevated ALA and / or PBG levels; The subject has a mutation associated with AHP; The subject has been diagnosed with renal impairment; and / or The subject has a reduced estimated glomerular filtration rate (eGFR), The method of claim 1.

5. The method of claim 1, wherein the reference level is the level of a healthy control or a previous level of the same subject.

6. 10. The method of claim 1, wherein the level of the biomarker is elevated by at least two-fold compared to the reference level of the biomarker.

7. 10. The method of claim 1, wherein the subject is being treated with a therapeutic agent that reduces expression of 5'-aminolevulinic acid synthase 1 (ALAS1).

8. The method of claim 7, wherein the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic agent.

9. 9. The method of claim 8, wherein the nucleic acid therapeutic agent is an RNAi agent or an antisense oligonucleotide.

10. 9. The method of claim 8, wherein the nucleic acid therapeutic agent is givosiran.

11. 8. The method of claim 7, wherein the subject is being treated with a second therapeutic agent.

12. 12. The method of claim 11, wherein the second therapeutic agent comprises a heme preparation, glucose, dextrose, or a combination thereof.

13. The method of claim 12, wherein the heme preparation is selected from the group consisting of hemin, heme alginate, and albumin-heme.

14. 10. The method of claim 1, wherein the level is determined in a subject sample selected from blood, plasma, serum, urine, or feces.

15. The method of claim 1, wherein the subject is suffering from one or more symptoms associated with AHP.

16. 16. A pharmaceutical composition comprising a therapeutic agent that reduces the expression of ALAS1 for use in treating AHP in a subject identified as suffering from AHP using the method defined in any of claims 1-15.

17. A pharmaceutical composition comprising a therapeutic agent that reduces the expression of ALAS1 for use in treating a human subject, wherein the subject has an elevated level of a biomarker compared to a reference level.

18. 18. The pharmaceutical composition of claim 17, wherein the biomarker is selected from the group consisting of KIM1, APLP1, MMP7, NGAL, CST3, and CHI3L1.

19. 18. The pharmaceutical composition of claim 17, wherein the biomarker is a renal injury biomarker.

20. 18. The pharmaceutical composition of claim 17, wherein the reference level is the level of a healthy control or a previous level in the same subject.

21. 18. The pharmaceutical composition of claim 17, wherein the level of the biomarker is elevated by at least two-fold compared to the reference level of the biomarker.

22. The pharmaceutical composition of claim 17, wherein the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic agent.

23. 23. The pharmaceutical composition of claim 22, wherein the nucleic acid therapeutic agent is an RNAi agent or an antisense oligonucleotide.

24. 24. The pharmaceutical composition of claim 23, wherein the nucleic acid therapeutic agent is givosiran.

25. The subject has not been diagnosed with AHP; The subject has not been diagnosed with porphyria; The subject does not meet the diagnostic criteria for AHP; The subject has a mutation associated with AHP; the subject does not have a mutation associated with AHP; The subject has recurrent acute attacks; The subject is a chronic high excretor; The subject has elevated ALA and / or PBG levels; The subject has a history of renal dysfunction; The subject has been diagnosed with renal impairment; or The subject has a reduced estimated glomerular filtration rate (eGFR), 18. The pharmaceutical composition of claim 17.

26. 18. The pharmaceutical composition of claim 17, wherein the subject is being treated with a second therapeutic agent.

27. 27. The pharmaceutical composition of claim 26, wherein the second therapeutic agent comprises a heme preparation, glucose, dextrose, or a combination thereof.

28. 28. The pharmaceutical composition of claim 27, wherein treatment with the second therapeutic agent is discontinued if the subject has an elevated level of the biomarker.

29. 18. The pharmaceutical composition of claim 17, wherein the level is determined in a subject sample selected from blood, plasma, serum, urine, or feces.

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