Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/08—Hepato-biliairy disorders other than hepatitis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/34—Genitourinary disorders
  • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

• the disclosure relates to biomarkers for acute hepatic porphyria (AHP). More specifically, the disclosure relates to renal injury biomarkers as biomarkers for AHP.
• AHP acute hepatic porphyria
• Acute hepatic porphyria is a group of rare genetic diseases caused by defects in enzymes in the heme biosynthesis 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.
• ALA delta- aminolevulinic acid
• PBG porphobilinogen
• a method of treating a human subject having acute hepatic porphyria comprises, for example, responsive to a determination of an elevated level of a biomarker (e.g., a renal injury biomarker) in a subject relative to a reference level, administering to the subject a therapeutic agent that reduces expression of 5 ’-aminolevulinic acid synthase 1 (ALAS1), thereby treating the subject.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter (CHE). In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• the biomarker e.g., renal injury biomarker
• the method comprises, for example, obtaining or having obtained a biological sample from the subject, performing or having performed an assay to determine the level of a biomarker (e.g., a renal injury biomarker) in the biological sample, and, if the subject has an elevated level of the biomarker (e.g., renal injury biomarker) relative to a reference value, administering to the subject a therapeutic agent that reduces expression of ALAS1.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• a method of treating a human subject having, or at risk of having, AHP can comprise providing a therapeutic agent that reduces the expression of ALAS1, detecting an elevated level of a biomarker (e.g., a renal injury biomarker) in the subject relative to a reference level, and administering the therapeutic agent to the subject if the level of the biomarker (e.g., renal injury biomarker) is greater than the reference level.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• a therapeutic agent that reduces expression of ALAS 1 for use in treating a human subject with AHP wherein the subject has an elevated level of a biomarker (e.g., a renal injury biomarker) as compared to a reference level.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• a therapeutic agent that reduces expression ALAS 1 for use in a method of treatment of a human subject with AHP, wherein the method comprises the step of determining if a patient has an elevated level of a biomarker (e.g., a renal injury biomarker) as compared to a reference level of the renal injury biomarker.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter.
• the subject has recurring acute attacks.
• the subject has an elevated level of ALA and/or PBG.
• the subject has a mutation associated with AHP.
• the subject does not have a mutation associated with AHP.
• the subject has a history of renal impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• an in vitro method of diagnosing AHP in a subject can comprise, for example, (a) determining the level of a biomarker (e.g., a renal injury biomarker) in a sample from the subject; (b) comparing the level of the biomarker (e.g., renal injury biomarker) determined in step (a) to a reference level of the biomarker (e.g., renal injury biomarker); and (c) assessing whether the subject suffers from AHP, wherein an increase in the level of the biomarker (e.g., renal injury biomarker) determined in step (a) as compared to the reference level of the biomarker (e.g., renal injury biomarker) is indicative of the subject suffering from AHP.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• a therapeutic agent that reduces expression of ALAS 1 for use in the treatment of AHP in a subject that has been identified as suffering from AHP using a method as defined herein.
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• a therapeutic agent that reduces expression of ALAS 1 for use in a method of treating AHP can comprise, for example, (a) determining the level of a biomarker (e.g., a renal injury biomarker) in a sample from the subject; (b) comparing the level of the biomarker (e.g., renal injury biomarker) determined in step (a) to a reference level of the biomarker (e.g., renal injury biomarker); (c) assessing whether the subject suffers from AHP, wherein an increase in the level of the biomarker (e.g., renal injury biomarker) determined in step (a) as compared to the reference level of the biomarker (e.g., renal injury biomarker) is indicative of the subject suffering from AHP; and (d) administering the therapeutic agent that reduces expression of ALAS Ito a subject that has been identified in step (c) as suffering from AHP.
• a biomarker e.g., a renal injury biomarker
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3L1.
• the biomarker is a renal injury biomarker.
• the renal injury biomarker is chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the therapeutic agent that reduces the expression of ALAS1 is a nucleic acid therapeutic.
• the nucleic acid therapeutic is an RNAi agent or an antisense oligonucleotide.
• the nucleic acid therapeutic is an RNAi agent described herein.
• the nucleic acid therapeutic is givosiran.
• the subject is a chronic high excreter. In some embodiments, the subject has recurring acute attacks. In some embodiments, the subject has not been diagnosed with AHP. In some embodiments, the subject has not been diagnosed as having a porphyria. In some embodiments, the subject does not meet the diagnostic criteria for AHP. In some embodiments, the subject has an elevated level 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 impairment. In some embodiments, the subject has been diagnosed with a renal injury. In some embodiments, the subject has a reduced estimated glomerular filtration rate (eGFR). In some embodiments, the subject is further suffering from one or more symptoms associated with AHP.
• eGFR estimated glomerular filtration rate
• the reference level is a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker
• the level of the biomarker is increased by 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., renal injury biomarker).
• the level is determined in a sample chosen from blood, plasma, serum, urine, or stool, from the subject.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the method further comprises discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker (e.g., renal injury biomarker).
• FIGS. 1A-1B includes graphs showing the difference in protein expression of plasma proteins between patients with AHP in two cohorts and healthy controls.
• FIGS. 2A-2C includes graphs showing kidney injury molecule- 1 (KIMI) levels in the different populations studied, KIMI levels in patients with a history of renal impairment, and KIMI levels plotted against estimated glomerular filtration rate (eGFR).
• KIMI kidney injury molecule- 1
• FIGS. 3A-3C includes graphs showing matrix metalloproteinase-7 (MMP7) levels in the different populations studied, MMP7 levels in patients with a history of renal impairment, and MMP7 levels plotted against eGFR.
• MMP7 matrix metalloproteinase-7
• FIGS. 4A-4C includes graphs showing neutrophil gelatinase-associated lipocalin (NGAL) levels in the different populations studied, NGAL levels in patients with a history of renal impairment, and NGAL levels plotted against eGFR.
• NGAL neutrophil gelatinase-associated lipocalin
• FIGS. 5A-5C includes graphs showing cystatin 3 (CST3) levels in the different populations studied, CST3 levels in patients with a history of renal impairment, and CST3 levels plotted against eGFR.
• CST3 cystatin 3
• FIGS. 6A-6C includes graphs showing chitinase-3-like protein 1 (CHI3L1) levels in the different populations studied, CHI3L1 levels in patients with a history of renal impairment, and CHI3L1 levels plotted against eGFR. DETAILED DESCRIPTION
• the inherited porphyrias are a family of disorders resulting from the deficient activity of specific enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway. Deficiency in the enzymes of the porphyrin pathway leads to insufficient heme production and to an accumulation of porphyrin precursors and porphyrins, which are toxic to tissue in high concentrations.
• AIP acute intermittent porphyria
• VP variegate porphyria
• HCP hereditary coproporphyria
• HCP hereditary coproporphyria
• ALA aminolevulinic acid
• ADP autosomal recessive ADP
• the acute attacks are characterized by autonomic, peripheral, and central nervous symptoms, including severe abdominal pain, hypertension, tachycardias, constipation, motor weakness, paralysis, and seizures. If not treated properly, quadriplegia, respiratory impairment, and death may ensue.
• Various factors including cytochrome P450-inducing drugs, dieting, and hormonal changes can precipitate acute attacks by increasing the activity of hepatic 5 ’-aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme of the heme biosynthetic pathway.
• ALAS1 hepatic 5 ’-aminolevulinic acid synthase 1
• the respective enzyme deficiencies result in hepatic production and accumulation of one or more substances (e.g., porphyrins and/or porphyrin precursors, e.g., ALA and/or PBG) that can be neurotoxic and can result in the occurrence of acute attacks.
• substances e.g., porphyrins and/or porphyrin precursors, e.g., ALA and/or PBG
• ALA and/or PBG neurotoxic and can result in the occurrence of acute attacks.
• AIP also referred to as porphobilinogen deaminase (PBGD) deficiency, or hydroxymethylbilane synthase (HMBS) deficiency
• PBGD porphobilinogen deaminase
• HMBS hydroxymethylbilane synthase
• AIP is an autosomal dominant disorder caused by mutations in the HMBS gene that result in reduced, e.g., half-normal activity of the enzyme.
• Diagnosis of porphyria can involve assessment of family history, assessment of porphyrin precursor levels in urine, blood, or stool, and/or assessment of enzyme activity and DNA mutation analysis.
• the differential diagnosis of porphyrias may involve determining the type of porphyria by measuring individual levels of porphyrins or porphyrin precursors (e.g., ALA, PBG) in the urine, feces, and/or plasma (e.g., by chromatography and fluorometry).
• samples must generally be obtained during an attack.
• the diagnosis of AIP can be confirmed by establishing that erythrocyte PBG deaminase activity is at 50% or less of the normal level.
• DNA testing for mutations may be another method of diagnosing AIP.
• AHP may present in the form of recurring acute attacks or chronic high excreters (CHE).
• CHE are a group of AHP patients that carry a genetic mutation and have elevated levels of ALA and PBG but are not experiencing acute attacks. Diagnosis of CHE can be more challenging. DNA testing for mutations may be required for patients and at-risk family members. Thus, the diagnosis of AHP, in particular, AIP typically requires confirmation by DNA testing and identification of a specific causative gene mutation (e.g., an HMBS mutation).
• Treatment of acute attacks typically requires hospitalization for control and treatment of acute symptoms, including, e.g., abdominal pain, seizures, dehydration/hyponatremia, nausea/vomiting, tachycardia/hypertension, urinary retention/ileus.
• abdominal pain may be treated, e.g., with narcotic analgesics
• seizures may be treated with seizure precautions and possibly medications (although many anti-seizure medications are contraindicated)
• nausea/vomiting may be treated, e.g., with phenothiazines
• tachycardia/hypertension may be treated, e.g., with beta blockers.
• Treatment may include withdrawal of unsafe medications, monitoring of respiratory function, as well as muscle strength and neurological status.
• Mild attacks may be treated with at least 300 g intravenous 10% glucose per day, although increasingly hemin is provided immediately. Severe attacks are typically treated as soon as possible with intravenous hemin (3-4 mg/kg daily for 4-14 days) and with IV glucose while waiting for the IV hemin to take effect. Typically, attacks are treated with IV hemin for 4 days and with IV glucose while waiting for administration of the IV hemin. Within 3-4 days following the start of hemin administration, there is usually clinical improvement accompanying by lowering of ALA and PBG levels.
• PANHEMATIN® or hemin for injection, previously known as hematin
• PANHEMATIN® is hemin derived from processed red blood cells (PRBCs) and is Protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand. Heme acts to limit the hepatic and/or marrow synthesis of porphyrin.
• hemin produces symptomatic improvement in patients with acute episodes of the hepatic porphyrias
• its action is likely due to the (feedback) inhibition of 6-aminolevulinic acid (ALA) synthase, the enzyme which limits the rate of the porphyrin/heme biosynthetic pathway.
• ALA 6-aminolevulinic acid
• PANHEMATIN® provides exogenous heme for the negative feedback inhibition of ALAS1, and thereby, decreases production of ALA and PBG. While patients generally respond well, its effect is relatively slow to normalize urinary ALA and PBG concentrations towards normal levels. As the intravenous hemin is rapidly metabolized, three to four infusions are usually necessary to effectively treat or prevent an acute attack.
• Givosiran is an AHP therapeutic agent that targets and causes degradation of ALAS 1 mRNA, reducing the production of neurotoxic intermediates ALA and PBG. Clinical trials for GIVLAARI® have demonstrated rapid and sustained reductions in ALA and PBG.
• AHP proteome associated with AHP
• CHE chronic high excreters
• APLP1 amyloid-like protein 1
• KIMI kidney injury molecule-1
• MMP7 matrix metalloproteinase-7
• KIMI and MP7 biomarkers for renal injury in some contexts.
• NGAL neutrophil gelatinase-associated lipocalin
• CST3 cystatin 3
• CHI3L1 chitinase-3 -like protein 1
• the disclosure provides protein biomarkers, including renal injury biomarkers, to evaluate a subject having, or at risk of having, AHP, e.g., a subject having an elevated level of ALA and/or PBG, or a subject with a mutation in an AHP associated gene.
• AHP e.g., a subject having an elevated level of ALA and/or PBG, or a subject with a mutation in an AHP associated gene.
• Early detection of an elevated biomarkers (e.g., renal injury biomarker) level can be used to prompt initiation of treatment of a subject with an agent that reduces the expression of ALAS 1 to treat AHP before the development of overt symptoms.
• the disclosure further provides for a method to select a therapeutic agent for treatment of a subject with AHP based on the level of biomarkers (e.g., renal injury biomarkers), including, but not limited to, APLP1, KIMI, MMP7, NGAL, CST3, and/or CHI3L1.
• biomarkers e.g., renal injury biomarkers
• APLP1, KIMI, MMP7, NGAL, CST3, and/or CHI3L1 include, but not limited to, APLP1, KIMI, MMP7, NGAL, CST3, and/or CHI3L1.
• Agents that reduce the expression of ALAS1 have been demonstrated in in pivotal trials to be effective in the treatment of AHP, including subjects that experience recurring acute attacks and CHE.
• the disclosure further provides diagnostic kits for detection of biomarkers (e.g., renal injury biomarkers), including, but not limited to, APLP1, KIMI, MMP7, NGAL, CST3, and/or CHI3L1, for use in the methods of disclosed herein.
• biomarkers e.g., renal injury biomarkers
• an element means one element or more than one element, e.g., a plurality of elements.
• the term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.
• the term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
• the term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context.
• the number of nucleotides in a nucleic acid molecule must be an integer.
• “at least 18 nucleotides of a 21- nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
• nucleotide overhang As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
• methods of detection can include determination that the amount of analyte present is below the level of detection of the method.
• an “AHP therapeutic agent” is understood as a therapeutic agent that reduces one or more symptom of AHP.
• the AHP therapeutic agent may be, for example, a therapeutic agent that reduces expression of ALAS1, or a therapeutic agent that stabilizes ALAS1.
• the AHP therapeutic agent prevents the production and accumulation of ALA and/or PBG.
• a “therapeutic agent that reduces expression of ALAS1” and the like as used herein is understood as a therapeutic agent that reduces levels of ALAS 1 RNA, ALAS1 protein, or both of ALAS1 RNA and ALAS1 protein.
• the therapeutic agent that reduces expression of ALAS1 is a therapeutic agent that promotes the degradation of an mRNA encoding ALAS 1 or inhibits the translation of an mRNA encoding ALAS1.
• agents include, but are not limited to, nucleic acid therapeutics, e.g., RNAi interference agents and antisense oligonucleotide agents. Such agents can typically inhibit 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.
• the agent is an iRNA.
• kidney injury biomarker or “kidney injury biomarker” is understood as at least a fragment of the sequence of a human polypeptide which has been previously associated with a renal or kidney injury.
• the injury may be a chronic renal or kidney injury or an acute renal or kidney injury.
• kidney injury molecule-1 or “KIMI” is understood as at least a fragment of the sequence of human KIMI polypeptide, for example, Accession Nos. NP_036338.2 (SEQ ID NO: 1), NP_001166864.1 (SEQ ID NO: 2), or NP_001295085.1 (SEQ ID NO: 3).
• KIMI can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• matrix metalloprotease-7 or “MMP7” is understood as at least a fragment of the sequence of human MMP7 polypeptide, for example, Accession No. NP_002414.1 (SEQ ID NO: 4).
• MMP7 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• NGAL neurotrophil gelatinase-associated lipocalin
• NGAL can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• diagnostic method e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• CST3 is understood as at least a fragment of the sequence of human CST3 polypeptide, for example, Accession Nos. NP_000090.1 (SEQ ID NO: 6) or NP_001275543.1 (SEQ ID NO: 7).
• CST3 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• CHI3L1 chitinase-3 -like protein 1
• CHI3L1 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• diagnostic method e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• APLP1 amyloid-like protein 1
• APLP1 can be specifically identified by any clinically acceptable diagnostic method, e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• diagnostic method e.g., antibody-based identification method, e.g., ELISA assay or immunoblotting; chromatography method; or single-molecule array (SIMOA).
• a “reference level” is understood as a predetermined level to which a level obtained from an assay, e.g., a biomarker level, e.g., a protein biomarker level, is compared.
• a reference level can be a control level determined for a healthy population, e.g., a population that does not have a disease or condition associated with a changed level of the biomarker and does not have a predisposition, e.g., genetic predisposition, to a disease or condition associated with a changed level of the biomarker.
• the population should be matched for certain criteria, e.g., age, gender.
• the reference level of the biomarker is a level from the same subject at an earlier time, e.g., before the development of symptomatic disease or before the start of treatment.
• samples are obtained from the subject at clinically relevant intervals, e.g., at intervals sufficiently separated in time that a change in the biomarker could be observed, e.g., at least three-month interval, at least a six-month interval, or at least a nine- month interval.
• any of the prior samples can act as a reference level.
• a “change as compared to a reference level” and the like is understood as a statistically or clinically significant change in the biomarker level, e.g., the change in the protein biomarker level, as compared to the reference level, is greater than the typical standard deviation of the assay method. Moreover, the change should be clinically relevant.
• the change as compared to a reference level can be determined as a percent change.
• a reference level is 100 pg/ml for biomarker X
• the level of biomarker X in the subject is 150 pg/ml
• the level of biomarker X in the subject is 300 pg/ml
• the level is increased by 300%.
• the level of biomarker X in the subject is 50 pg/ml
• the level is decreased by 50%.
• the change as compared to a reference level is increased by at least 50%.
• the change as compared to a reference level is increased by at least 100%, at least 200%, or at least 300%. In certain embodiments, the change as compared to a reference sample is decreased by at least 25%. In certain embodiments, the change as compared to a reference sample is decreased by at least 50%.
• biological fluids include blood, serum, serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, stool, saliva, and the like.
• Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be liver tissue or be derived from the liver.
• a “biological sample from a subject” can refer to blood or blood derived serum or plasma from the subject.
• the fluid is substantially free of cells, e.g., is free of cells.
• administering a therapeutic agent is understood as providing a therapeutic agent to a subject.
• the therapeutic agent is provided at an appropriate dosage and by a route of administration for the agent as provided, for example, by the label of the therapeutic agent.
• ALAS1 also known as ALAS-1; 6-aminolevulinate synthase 1; 6- ALA synthase 1; 5 ’-aminolevulinic acid synthase 1; ALAS-H; ALASH; ALAS-N; ALAS3; EC2.3.1.37; 5-aminolevulinate synthase, nonspecific, mitochondrial; ALAS; MIG4; GTTHUMP00000212619; GTTHUMP00000212620; 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.
• ALAS 1 catalyzes the condensation of glycine with succinyl-CoA to form 6-aminolevulinic acid (ALA).
• the human ALAS1 gene is expressed ubiquitously, is found on chromosome 3p21.1 and typically encodes a sequence of 640 amino acids.
• the ALAS-2 gene which encodes an isozyme, is expressed only in erythrocytes, is found on chromosome Xp 11.21, and typically encodes a sequence of 550 amino acids.
• an “ALAS 1 protein” means any protein variant of ALAS 1 from any species (e.g., human, mouse, non-human primate), as well as any mutants and fragments thereof that retain an ALAS 1 activity.
• an “ALAS 1 transcript” refers to any transcript variant of ALAS1, from any species (e.g., human, mouse, non-human primate).
• a sequence of a human ALAS1 mRNA transcript can be found, for example, at NM_000688.4 (SEQ ID NO: 11).
• the level of the mature encoded ALAS 1 protein is regulated by heme: high levels of heme down-regulate the mature enzyme in mitochondria while low heme levels up-regulate. Multiple alternatively spliced variants, encoding the same protein, have been identified.
• RNAi RNA agent
• RNAi agent refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway.
• RISC RNA-induced silencing complex
• an iRNA as described herein effects 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 the ALAS 1 protein.
• target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ALAS 1 gene, including mRNA that is a product of RNA processing of a primary transcription product.
• the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion.
• the target sequence will generally be from 9-36 nucleotides in length, e.g., 15- 30 nucleotides in length, including all sub-ranges therebetween.
• the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-
• 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, 19-
• nucleic acid therapeutic agent is understood as a therapeutic agent comprising a sufficient length of nucleotides to specifically hybridize to a target sequence in a target nucleic acid in a cell such that the hybridization reduces levels of a protein encoded by the target nucleic acid, e.g., by inhibiting translation or promoting sequence specific degradation of the target nucleic acid.
• exemplary nucleic acid therapeutic agents include RNAi agents and antisense oligonucleotide agents.
• antisense polynucleotide agent refers to an agent comprising a single- stranded oligonucleotide that specifically binds to the target nucleic acid molecules via hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) and inhibits the expression of the targeted nucleic acid by an antisense mechanism of action, e.g., by RNase H.
• hydrogen bonding e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding
• an antisense agent is a nucleic acid therapeutic that acts by reducing the expression of a target gene, thereby reducing the expression of the polypeptide encoded by the target gene.
• exemplary antisense agents that reduce or inhibit the expression of ALAS1 include an antisense strand that comprises, or consists of, the antisense sequence of AD-60489, AD-60519, AD-61193, or AD-60819 (or a corresponding unmodified antisense sequence) (see, e.g., U.S. Patent No. 10,119,143 which is incorporated by reference in its entirety).
• RNAi agent that reduces the expression of ALAS1, givosiran, is provided, for example, in U.S. Patent No. 10,119,143, titled “Compositions and methods for inhibiting expression of the ALAS1 gene,” filed April 4, 2016, incorporated herein by reference in its entirety for all purposes.
• 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-methyladenosine- 3 ’-phosphate, 2'-O-methylcytidine-3 ’-phosphate, 2'-O-methylguanosine-3’ -phosphate, and 2'- O-methyluridine-3’ -phosphate, respectively; Af, Cf, Gf, and Uf are 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 phosphorothi
• treatment is understood as administration of a therapeutic agent to reduce the rate of progression of a disease or condition or to reduce at least one sign or symptom in a subject suffering from a disease.
• a sign of a disease can be a change in a biomarker from a healthy reference level prior to the development of overt symptoms of the disease. Natural history studies and clinical trials of AHP have demonstrated disease progression in the absence of treatment.
• detection of a protein As used herein, “detection of a protein”, “detection of a biomarker”, and the like are understood as detection of a protein, or a sufficiently large fragment of the protein, to determine the identity of the protein by the method used, e.g., immunological method, chromatography method.
• detection of a protein can include detection of a one or more isoforms of a protein present in a subject when no distinction is made among the various isoforms.
• the detection method is a clinically accepted or validated method.
• Plasma proteomics and the identification of minimally invasive biomarkers is emerging as an integral part of modern drug discovery and clinical development.
• the proteomics approach demonstrated that 212 plasma proteins of 1196 proteins studied were significantly different between healthy controls and patients with AHP. Of those 212 plasma proteins, three proteins with the greatest effect size difference between AHP patients and healthy controls include APLP1, KIMI, and MMP7 (FIGS. 1A-1B). Two of the three proteins with the greatest effect size between AHP patients and healthy controls (KIMI, MMP7) are biomarkers of renal injury. Additional renal injury biomarkers were explored based on measured plasma proteins and previous characterization of plasma levels and renal injury. Three additional renal injury biomarkers (NGAL, CST3, CHI3L1) showed significant elevations in patients with AHP.
• GFR Glomerular filtration rate
• renal injury biomarker levels are elevated in AHP patients.
• renal injury protein levels may serve as a potential biomarker in other aspects of AHP disease. If levels of renal injury biomarkers increase during AHP early disease development, the renal injury biomarkers may serve as a prognostic indicator for the incidence of symptomatic disease, e.g., an acute attack as compared to CHE. Additionally, the correlation of renal injury biomarker levels with eGFP indicates that the renal injury biomarker levels may correlate with severity of the AHP disease. Eastly, renal injury biomarkers may potentially play an important role for distinguishing between effectiveness of various treatments. The present study was based on patients with AHP recurring acute attacks as well as CHE. Therefore, the presence of elevated renal injury biomarkers is not limited to patients exhibiting exclusively an acute attack porphyria.
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APEP1, MMP7, NGAE, CST3, or CHI3El.
• the biomarker comprises KIMI.
• the biomarker comprises APEP1.
• the biomarker comprises MMP7.
• the biomarker comprises NGAE.
• the biomarker comprises CST3.
• the biomarker comprises CHI3E1.
• the biomarker is a renal injury biomarker chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3E1.
• the levels of a plurality of biomarkers are determined.
• the biomarkers comprise KIMI and APLP1.
• the biomarkers comprise KIMI and MMP7.
• the biomarkers comprise KIMI and NGAL.
• the biomarkers comprise KIMI and CST3.
• the biomarkers comprise KIMI and CHI3L1.
• the biomarkers comprise APLP1 and MMP7.
• the biomarkers comprise APLP1 and NGAL.
• the biomarkers comprise APLP1 and CST3.
• the biomarkers comprise APLP1 and CHI3L1.
• the biomarkers comprise MMP7 and NGAL. In some embodiments, the biomarkers comprise MMP7 and CST3. In some embodiments, the biomarkers comprise MMP7 and CHI3L1. In some embodiments, the biomarkers comprise NGAL and CST3. In some embodiments, the biomarkers comprise NGAL and CHI3L1. In some embodiments, the biomarkers comprise CST3 and CHI3L1.
• the biomarkers comprise KIMI, MMP7, and APLP1. In some embodiments, the biomarkers comprise KIMI, MMP7, and NGAL. In some embodiments, the biomarkers comprise KIMI, MMP7, and CST3. In some embodiments, the biomarkers comprise KIMI, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, and NGAL.
• the biomarkers comprise APLP1, MMP7, and CST3. In some embodiments, the biomarkers comprise APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, and CST3. In some embodiments, the biomarkers comprise APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the
• the biomarkers comprise KIMI, APLP1, MMP7, and NGAL. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, NGAL, and CST3.
• the biomarkers comprise KIMI, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1,
• the biomarkers comprise KIMI, APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, CST3, and CHI3L1.
• the biomarkers comprise KIMI, APLP1, MMP7, NGAL, CST3, and CHI3L1.
• the biomarkers further comprise KIMI.
• the biomarkers further comprise APLPL
• the biomarkers further comprise MMP7.
• the biomarkers further comprise NGAL.
• the biomarkers further comprise CST3.
• the biomarkers further comprise CHI3L1.
• Biomarkers e.g., renal injury biomarkers, to determine when a subject with a predisposition to AHP, e.g., a subject with a genetic predisposition or a subject with elevated ALA and/or PBG, may be treated with an agent that reduces the expression of ALAS1.
• the biomarker is chosen from one or more (e.g., 2, 3, 4, 5, or all) of KIMI, APLP1, MMP7, NGAL, CST3, or CHI3Ll.
• the biomarker comprises KIMI.
• the biomarker comprises APLP1.
• the biomarker comprises MMP7.
• the biomarker comprises NGAL.
• the biomarker comprises CST3.
• the biomarker comprises CHI3L1.
• the biomarker is a renal injury biomarker chosen from one or more (e.g., 2, 3, 4, or all) of KIMI, MMP7, NGAL, CST3, or CHI3L1.
• the levels of a plurality of biomarkers are determined.
• the biomarkers comprise KIMI and APLP1.
• the biomarkers comprise KIMI and MMP7.
• the biomarkers comprise KIMI and NGAL.
• the biomarkers comprise KIMI and CST3.
• the biomarkers comprise KIMI and CHI3L1.
• the biomarkers comprise APLP1 and MMP7.
• the biomarkers comprise APLP1 and NGAL.
• the biomarkers comprise APLP1 and CST3.
• the biomarkers comprise APLP1 and CHI3L1.
• the biomarkers comprise MMP7 and NGAL. In some embodiments, the biomarkers comprise MMP7 and CST3. In some embodiments, the biomarkers comprise MMP7 and CHI3L1. In some embodiments, the biomarkers comprise NGAL and CST3. In some embodiments, the biomarkers comprise NGAL and CHI3L1. In some embodiments, the biomarkers comprise CST3 and CHI3L1.
• the biomarkers comprise KIMI, MMP7, and APLP1. In some embodiments, the biomarkers comprise KIMI, MMP7, and NGAL. In some embodiments, the biomarkers comprise KIMI, MMP7, and CST3. In some embodiments, the biomarkers comprise KIMI, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, and NGAL.
• the biomarkers comprise APLP1, MMP7, and CST3. In some embodiments, the biomarkers comprise APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, and CST3. In some embodiments, the biomarkers comprise APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise NGAL, CST3, and CHI3L1. In some embodiments, the
• the biomarkers comprise KIMI, APLP1, MMP7, and NGAL. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, NGAL, and CST3.
• the biomarkers comprise KIMI, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1,
• the biomarkers comprise KIMI, APLP1, MMP7, NGAL, and CST3. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, NGAL, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, MMP7, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, APLP1, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise KIMI, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, CST3, and CHI3L1. In some embodiments, the biomarkers comprise APLP1, MMP7, NGAL, CST3, and CHI3L1.
• the biomarkers comprise KIMI, APLP1, MMP7, NGAL, CST3, and CHI3L1.
• the biomarkers further comprise KIMI.
• the biomarkers further comprise APLP1.
• the biomarkers further comprise MMP7. In some embodiments, the biomarkers further comprise NGAL. In some embodiments, the biomarkers further comprise CST3. In some embodiments, the biomarkers further comprise CHI3L1.
• the subject is routinely monitored for an increase in biomarker (e.g., renal biomarker) levels as compared to a reference level, either a population control or a biomarker (e.g., renal biomarker) level for the same subject.
• An increase in the biomarker (e.g., renal biomarker) in the subject is an indicator treatment of the subject, e.g., with an agent that reduces the expression of ALAS1 should be initiated.
• the subject is also routinely monitored for the development of a sign or symptom of AHP.
• the subject is also monitored for the level of one or more of ALA and PBG, wherein an increase in the level of a biomarker (e.g., renal biomarker) as compared to a reference level when the beta coefficient is positive is indicative of worsening AHP, and a decrease in the level of a biomarker (e.g., renal biomarker) as compared to a reference level when the beta coefficient is negative is indicative of worsening AHP.
• a biomarker e.g., renal biomarker
• Monitoring of biomarker (e.g., renal biomarker) levels can also be used to determine if AHP is progressing in the subject, where an increase in the level of the biomarker (e.g., renal biomarker) in the subject is indicative of progressive AHP. Progression can be monitored by further determining the level of one or more of ALA and PBG, wherein an increase in the level of a biomarker (e.g., renal biomarker) as compared to a reference level when the beta coefficient is positive is indicative of worsening AHP, and a decrease in the level of a biomarker (e.g., renal biomarker) as compared to a reference level when the beta coefficient is negative is indicative of worsening AHP.
• ALA and PBG wherein an increase in the level of a biomarker (e.g., renal biomarker) as compared to a reference level when the beta coefficient is positive is indicative of worsening AHP, and a decrease in the level of a biomarker (e.g.
• the disclosure provides the steps of measuring one or more biomarker (e.g., protein biomarker) levels in a subject and comparison of a biomarker level to its corresponding reference level to determine if there is a difference between the biomarker level and its corresponding reference level.
• biomarker e.g., protein biomarker
• the method for determining the level of the biomarker in the sample and the method by which the reference level was determined for the reference level should be the same.
• the change from the reference level may be a change in a defined concentration of a biomarker in a sample, e.g., pg/ml of sample.
• the change can be a relative amount, a change in percent of the reference sample, e.g., at least 150% or at least 200% of the reference sample.
• the change in the level should be statistically significant. Methods to determine biomarker levels are typically performed in vitro, often in a clinical lab setting when used for diagnostic methods and methods used to select a treatment for a subject.
• kits are available to determine the level of some biomarkers, e.g., NT-proBNP and troponin I. Levels of these markers can be performed in clinical laboratories using commercially available diagnostic tests, e.g., using chemiluminescence assays (Roche Diagnostic Cobas, Indianapolis, IN, USA for NT-proBNP; Siemens Centaur XP, Camberley, Surrey, UK for troponin I). In such cases, the reference level of the biomarker, and in embodiments an appropriate control, can be provided by the kit manufacturer.
• a sufficient interval to allow for a change in biomarker level needs to be provided.
• Changes in renal injury biomarker levels were observed between day 0 and one or more subsequent time point, both in control subjects with increasing levels of the renal injury biomarker, and in treated subjects with decreasing levels.
• a sufficient interval for a change in renal injury biomarker level may be determined for each renal injury biomarker. It is expected that short intervals, e.g., 6 months, 3 months, 2 months, 1 month or even less than one month, can be sufficient to observe a change in renal injury biomarker level in a subject, depending on the particular biomarker, disease state, and rate of progression in the subject.
• the methods disclosed herein can further include monitoring subjects for one or more signs or symptoms indicative of AHP or progression of AHP including, but not limited to, abdominal pain, limb, back, or chest pain, nausea, vomiting, confusion, anxiety, seizures, constipation, diarrhea, dark reddish urine, or any combination thereof.
• Comorbidities indicative of AHP or AHP progression that may be monitored include, for example, hypertension, chronic kidney disease, and combinations thereof.
• the development or progression of one or more of the signs or symptoms, in the context of an elevated renal injury biomarker level as compared to a reference level, is further diagnostic of AHP.
• the methods described herein involve use of a nucleic acid therapeutic agent (e.g., an RNAi agent) that reduces expression of ALAS1.
• a nucleic acid therapeutic agent e.g., an RNAi agent
• expression of an ALAS1 gene is reduced or inhibited using an ALAS 1- specific iRNA.
• compositions and methods that effect the RNA induced silencing complex (RlSC)-mediated cleavage of RNA transcripts of the ALAS 1 gene, such as in a cell or in a subject (e.g., in a mammal, such as a human subject).
• RlSC RNA induced silencing complex
• the iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS 1 gene (e.g., a mouse or human ALAS 1 gene) (also referred to herein as an “ALAS 1- specific iRNA”).
• ALAS 1 gene e.g., a mouse or human ALAS 1 gene
• iRNAs encompass a dsRNA having an RNA strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS 1 gene (e.g., a human variant 1 or 2 of an ALAS1 gene) (also referred to herein as a “ALAS 1- specific iRNA”).
• ALAS 1 gene e.g., a human variant 1 or 2 of an ALAS1 gene
• ALAS 1- specific iRNA also referred to herein as a “ALAS 1- specific iRNA”.
• the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human ALAS1.
• the human ALAS1 has the sequence of NM_000688.4 (SEQ ID NO: 11).
• the human ALAS1 has the sequence of NM_199166.1 (SEQ ID NO: 12).
• the antisense sequence of the iRNA targets within the region 871 to 895 (plus or minus 5, 4, 3, 2, or 1 nucleotides in either or both directions on the 5’ and/or 3’ end) on the ALAS1 transcript NM_000688.4.
• the antisense sequence targets the nucleotides 871 to 893, 871 to 892, or 873 to 895 on the ALAS1 transcript NM_000688.4.
• 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 on the ALAS1 transcript NM_000688.4.
• a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS 1 comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS 1 RNA transcript, which antisense strand comprises at least 15 (e.g., at least 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides differing by no more than 3, 2 or 1 nucleotides from the sequence of UAAGAUGAGACACUCUUUCUGGU (SEQ ID NO: 13) or UAAGAUGAGACACUCTUUCUGGU (SEQ ID NO: 14).
• dsRNA double-stranded ribonucleic acid
• the antisense strand comprises the sequence of UAAGAUGAGACACUCUUUCUGGU (SEQ ID NO: 13) or UAAGAUGAGACACUCTUUCUGGU (SEQ ID NO: 14).
• the sense strand comprises the sequence of CAGAAAGAGUGUCUCAUCUUA (SEQ ID NO: 15).
• one or more nucleotides of the antisense strand and/or sense strand are modified as described herein.
• the dsRNA is effective to suppress the liver level of ALAS 1 mRNA, e.g., to achieve silencing of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% (e.g., such that ALAS1 mRNA levels are decreased 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 liver ALAS1 mRNA, e.g., the level in an untreated individual or group of individuals, e.g., an individual or group of individuals treated with PBS only).
• a control level of liver ALAS1 mRNA e.g., the level in an untreated individual or group of individuals, e.g., an individual or group of individuals treated with PBS only.
• the dsRNA is effective to suppress the circulating level of ALAS1 mRNA, e.g., to achieve silencing of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% (e.g., such that ALAS1 mRNA levels are decreased 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 prior to treatment with the dsRNA, or the level in an untreated individual or group of individuals).
• a control level of circulating ALAS1 mRNA e.g., the level prior to treatment with the dsRNA, or the level in an untreated individual or group of individuals.
• the iRNA molecules featured herein can include naturally occurring nucleotides or can include at least one modified nucleotide.
• the at least one modified nucleotide include one or more of a modification on the nucleotide chosen from the group consisting of a locked nucleic acid (LNA), an acyclic nucleotide, a hexitol or hexose nucleic acid (HNA), a cyclohexene nucleic acid (CeNA), 2’ -methoxyethyl, 2’-O-alkyl, 2’-O-allyl, 2’- C- allyl, 2’-fluoro, 2’-deoxy, 2’-hydroxyl, or any combination thereof.
• LNA locked nucleic acid
• HNA hexitol or hexose nucleic acid
• CeNA cyclohexene nucleic acid
• the at least one modified nucleotide includes, but is not limited to a 2'-O-methyl modified nucleotide, 2’-fluoro modified nucleotide, a nucleotide having a 5'-phosphorothioate group, and a terminal nucleotide linked to a ligand, e.g., an N-acetylgalactosamine (GalNAc) or a cholesteryl derivative.
• a ligand e.g., an N-acetylgalactosamine (GalNAc) or a cholesteryl derivative.
• the modified nucleotide may be chosen from the group of: a 2'-deoxy- 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an acyclic nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
• the region of complementarity is at 5 least 17 nucleotides in length.
• 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 no more than 30 nucleotides in length.
• At least one strand comprises a 3’ overhang of at least 1 nucleotide.
• the antisense strand comprises a 3’ overhang of at least 1 nucleotide.
• 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 2 nucleotides.
• an iRNA as described herein targets a wildtype ALAS 1 RNA transcript variant, and in another embodiment, the iRNA targets a mutant transcript (e.g., an ALAS1 RNA carrying an allelic variant).
• a mutant transcript e.g., an ALAS1 RNA carrying an allelic variant.
• an iRNA featured in the disclosure can target a polymorphic variant, such as a single nucleotide polymorphism (SNP), of ALAS1.
• the iRNA targets both a wildtype and a mutant ALAS1 transcript.
• the iRNA targets a particular transcript variant of ALAS1 (e.g., human ALAS1 variant 1).
• the iRNA agent targets multiple transcript variants (e.g., both variant 1 and variant 2 of human ALAS1).
• an iRNA featured in the disclosure targets a non-coding region of an ALAS1 RNA transcript, such as the 5’ or 3’ untranslated region of a transcript.
• an iRNA as described herein is in the form of a conjugate, e.g., a carbohydrate conjugate, which may serve as a targeting moiety and/or ligand, as described herein.
• the conjugate is attached to the 3’ end of the sense strand of the dsRNA.
• the conjugate is attached via a linker, e.g., via a bivalent or trivalent branched linker.
• the conjugate comprises one or more N-acetylgalactosamine (GalNAc) derivatives.
• GalNAc N-acetylgalactosamine
• the conjugate targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte.
• the GalNAc derivatives can be attached via a linker, e.g., a bivalent or trivalent branched linker.
• the RNAi agent is attached to the carbohydrate conjugate via a linker.
• the dsRNA comprises a duplex region which is 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 no more than 26 nucleotides in length.
• the antisense strand consists of the sequence of usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsu (SEQ ID NO: 17). In some embodiments, the sense strand consists of the sequence of csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 16).
• the sense strand consists of the sequence of csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 16), and the antisense strand consists of the sequence of usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsu (SEQ ID NO: 17).
• the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, wherein said dsRNA is in the form of a conjugate having the structure of:
• the dsRNA comprises a duplex region which is 21 nucleotide pairs in length.
• the antisense strand comprises a 3’ overhang of two nucleotides.
• nucleic acid therapeutics that reduce that reduce the expression of ALAS1 are disclosed in International Application Publication Nos. WO2013/155204 and W02015/051318, 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 Nos. US2013/0281511, US2015/0111841, US2016/0115476, US2016/0244766, US2018/0037886, US2019/0144870, US2019/0218549, US2020/0181614, and US2021/0087558, the contents of each of the aforesaid publications are incorporated by reference in their entirety.
• compositions e.g., a pharmaceutical composition, that includes one or more of the iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle.
• the composition is used for inhibiting the expression of an ALAS 1 gene in an organism, generally a human subject.
• the composition is used for treating a porphyria, e.g., AHP, e.g., AIP.
• the iRNA e.g., dsRNA
• the unbuffered solution is saline or water, e.g., water for injection.
• the pharmaceutical composition comprises the iRNA and water for injection.
• the composition comprises about 100 to 300 mg/mL, e.g., 200 mg/mL, of the iRNA.
• the composition has a pH of 6.0-7.5, e.g., about 7.0.
• the composition is for subcutaneous injection.
• the pharmaceutical composition is packaged in a container (e.g., a glass vial, e.g., a 2 mL glass vial,) at a volume of about 0.3 to 1 mL, e.g., 0.55 mL.
• the iRNA e.g., dsRNA is administered with a buffer solution.
• the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
• the buffer solution is phosphate buffered saline (PBS).
• the iRNA e.g., dsRNA
• the iRNA is targeted to hepatocytes.
• the composition is administered intravenously. In embodiments of the pharmaceutical compositions described herein, the composition is administered subcutaneously.
• the dsRNA is administered monthly at a dose of 0.01 mg/kg to 5 mg/kg or 1 mg/kg to 2.5 mg/kg bodyweight of the subject. In some embodiments, the dsRNA is administered monthly at a dose of 2.5 mg/kg bodyweight of the subject.
• a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand) that targets the iRNA (e.g., dsRNA) to hepatocytes.
• a ligand e.g., a GalNAc ligand
• a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand), and the pharmaceutical composition is administered subcutaneously.
• the ligand targets the iRNA (e.g., dsRNA) to hepatocytes.
• a pharmaceutical composition e.g., a composition described herein, includes a lipid formulation.
• the RNAi agent is in an LNP formulation, e.g., a MC3 formulation.
• the LNP formulation targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte.
• the lipid formulation is a LNP 11 formulation.
• the composition is administered intravenously.
• the pharmaceutical composition is formulated for administration according to a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week.
• the administration of the pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.
• a composition containing an iRNA featured in the disclosure is administered with a non-iRNA therapeutic agent, such as an agent known to treat a porphyria (e.g., AIP), or a symptom of a porphyria (e.g., pain).
• a composition containing an iRNA featured in the disclosure e.g., a dsRNA targeting AIP, is administered along with a non-iRNA therapeutic regimen, such as hemin or glucose (e.g., glucose infusion (e.g., IV glucose)).
• an iRNA featured in the disclosure can be administered before, after, or concurrent with glucose, dextrose, or a similar treatment that serves to restore energy balance (e.g., total parenteral nutrition).
• An iRNA featured in the disclosure can also be administered before, after, or concurrent with the administration of a heme product (e.g., hemin, heme arginate, or heme albumin), and optionally also in combination with a glucose (e.g. IV glucose) or the like.
• a heme product e.g., hemin, heme arginate, or heme albumin
• glucose e.g. IV glucose
• IV glucose administered for the treatment of a porphyria is administered intravenously (IV).
• IV glucose administration of glucose intravenously
• alternative embodiments in which glucose is administered by other means are also encompassed.
• an ALAS 1 iRNA is administered to a patient, and then the non- iRNA agent or therapeutic regimen (e.g., glucose and/or a heme product) is administered to the patient (or vice versa).
• the non- iRNA agent or therapeutic regimen e.g., glucose and/or a heme product
• an ALAS1 iRNA and the non-iRNA therapeutic agent or therapeutic regimen are administered at the same time.
• the subject may have recurring acute attacks of AHP.
• the subject may be a chronic high excreter.
• the subject has not been diagnosed as having a porphyria. In some embodiments, the subject has not been diagnosed with AHP. For instance, the subject may not have been determined by a health care professional to meet one or both of a clinically determinative level of ALA and/or PBG and a mutation associated with AHP.
• the subject does not meet the diagnostic criteria for AHP. For instance, the subject may not meet one or both of (i) having a clinically determinative level of ALA and/or PBG and (ii) having a mutation associated with AHP.
• the subject has an elevated level of ALA and/or PBG.
• Elevated levels of ALA may be, for example, more than 35 pmol/L in a biological sample or more than 60 pmol/d in a 24-hour period.
• Elevated levels of PBG may be, for example, more than 8.8 pmol/d in a 24-hour period.
• the subject has a mutation associated with AHP.
• the subject may have a specific AHP causative gene mutation (e.g., an HMBS mutation).
• the subject does not have a mutation associated with AHP.
• the subject may not have a specific AHP causative gene mutation (e.g., an HMBS mutation).
• the subject has a history of renal impairment.
• the subject may have previously been diagnosed with a renal impairment.
• the subject may have recovered from the renal impairment.
• the subject may be suffering from the renal impairment.
• the subject has been diagnosed with a renal injury.
• the subject may have been diagnosed with chronic kidney disease or an acute kidney injury.
• the subject has a reduced estimated glomerular filtration rate (eGFR).
• the subject may have a stage 1, stage 2, stage 3, stage 4, or stage 5 eGFR.
• Stage 1 eGFR of 90 or higher
• Stage 2 eGFR between 60 and 89
• Stage 3 eGFR between 30 and 59
• Stage 4 eGFR between 15 and 29
• Stage 5 is a sign of kidney failure associated with less than 15% kidney function.
• the level of the biomarker (e.g., renal injury biomarker) in the subject is increased by at least 2-fold, for example, 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, 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 the reference level of the renal injury biomarker.
• the reference level may be a healthy control level or an earlier level in the same subject.
• the level of the biomarker e.g., renal injury biomarker may be determined in a subject sample selected from blood, plasma, serum, urine, or stool.
• the subject is being treated with a second therapeutic agent.
• the second therapeutic agent may be a therapeutic agent for AHP.
• the second therapeutic agent comprises a heme product (e.g., hemin, heme arginate, or heme albumin), glucose (e.g. IV glucose), dextrose, or combinations thereof.
• the second therapeutic agent may be a therapeutic agent for renal injury.
• the second therapeutic agent may be a therapeutic agent for chronic renal injury or acute renal injury.
• the second therapeutic agent may be a therapeutic agent for a symptom of AHP.
• the second therapeutic agent may be a therapeutic agent for a comorbidity of AHP.
• the method may further comprise discontinuation of treatment with the second therapeutic agent when the subject has the elevated level of the biomarker, e.g., renal injury biomarker.
• the biomarker e.g., renal injury biomarker.
• the subject is further suffering from one or more symptoms associated with AHP.
• the subject may be suffering from abdominal pain, limb, back, or chest pain, nausea, vomiting, confusion, anxiety, seizures, constipation, diarrhea, dark reddish urine, or any combination thereof.
• the subject is further suffering from one or more comorbidities associated with AHP.
• the subject may be suffering from hypertension, chronic kidney disease, or both.
• Example 1 Proteins Identified that Differ in AHP Patients Relative to Healthy Controls
• proteomics assay plasma levels were analyzed to find a set of biomarkers indicative of AHP disease compared to healthy controls, symptomatic disease compared to CHE, and severity of AHP disease.
• the patients from each of cohort 1 and cohort 2 were compared to healthy controls.
• a total of 212 plasma proteins were identified that differ in AHP patients relative to healthy controls.
• the 212 plasma proteins are plotted in the graphs of FIG. 1A (cohort 1) and FIG. IB (cohort 2).
• KIMI kidney injury molecule- 1
• biomarkers including, for example, KIMI, APLP1, and MMP7, may aid in diagnosing and managing kidney disease in patients with AHP.
• biomarkers of Renal Injury are Elevated in AHP Patients and CHE Relative to Healthy Controls
• Biomarkers of renal injury were found to be significantly different between AHP patients and healthy controls in Example 1. Other biomarkers of renal injury were further studied. The results for five of these proteins are shown in the graphs of FIGS. 2A-2C, 3A- 3C, 4A-4C, 5A-5C, and 6A-6C, respectively.
• Plasma protein levels of the renal injury biomarkers were measured in the AHP patients from cohort 1 and cohort 2, as well as CHE patients, and compared to healthy controls.
• KIMI and MMP7 are both significantly elevated in AHP patients from both cohorts and CHE relative to healthy control samples (p-values ⁇ 0.01; FIGS. 2A and 3A, respectively).
• Renal injury biomarkers were selected to be explored based on measured OLINK® proteins from Example 1 and previous characterization of plasma levels and renal injury. Renal injury biomarkers neutrophil gelatinase-associated lipocalin (NGAL), cystatin 3 (CST3) and chitinase- 3 -like protein 1 (CHI3L1) showed significant elevations in patients with AHP (FIGS. 4A, 5A, and 6A, respectively). AHP patients with a diagnosis of renal disease demonstrated significantly higher levels of each of these biomarkers than patients without such a diagnosis (p-values ⁇ 0.01) (FIGS. 4B, 5B, and 6B, respectively).
• neutrophil gelatinase-associated lipocalin NGAL
• CST3 cystatin 3
• CHI3L1 chitinase- 3 -like protein 1
• biomarkers of renal injury were found to be elevated in AHP patients relative to healthy controls
• KIMI, MMP7, and CST3 were also found to be elevated in CHE patients
• KIMI, MMP7, NGAL, CST3, and CHI3L1 were further elevated in patients with a previous diagnosis of renal injury and were moderately correlated with eGFR.
• renal injury biomarkers may aid in diagnosing and managing kidney disease in patients with AHP suffering from recurrent acute attacks as well as chronic high excreters. These biomarkers may be useful for diagnosis and monitoring of renal injury in AHP patients.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Cell Biology (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Virology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biophysics (AREA)
• Gastroenterology & Hepatology (AREA)
• Plant Pathology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Related Child Applications (1)

Publications (1)

Family

ID=84331708

Family Applications (1)

Country Status (5)

Cited By (1)

Citations (3)

• 2022
  • 2022-10-04 WO PCT/US2022/077511 patent/WO2023060064A1/en not_active Ceased
  • 2022-10-04 JP JP2024520992A patent/JP2024537202A/ja active Pending
  • 2022-10-04 EP EP22802413.9A patent/EP4413378A1/en active Pending
  • 2022-10-04 TW TW111137690A patent/TW202329984A/zh unknown
• 2024
  • 2024-04-01 US US18/623,281 patent/US20240360454A1/en active Pending

Patent Citations (18)

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events