WO2021250530A1 - Procédé rapide de génotypage de variants sting chez des individus humains - Google Patents

Procédé rapide de génotypage de variants sting chez des individus humains Download PDF

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WO2021250530A1
WO2021250530A1 PCT/IB2021/054949 IB2021054949W WO2021250530A1 WO 2021250530 A1 WO2021250530 A1 WO 2021250530A1 IB 2021054949 W IB2021054949 W IB 2021054949W WO 2021250530 A1 WO2021250530 A1 WO 2021250530A1
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sting
fragment
variant
seq
gene
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PCT/IB2021/054949
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English (en)
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Ritika RAINA
Abhisek CHATTERJEE
Debjani CHAKRABORTY
Sandip Kumar MIDDYA
Arjun SURYA
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Curadev Pharma Pvt. Ltd.,
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Priority claimed from GBGB2008579.1A external-priority patent/GB202008579D0/en
Application filed by Curadev Pharma Pvt. Ltd., filed Critical Curadev Pharma Pvt. Ltd.,
Publication of WO2021250530A1 publication Critical patent/WO2021250530A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the “Stimulator of Interferon Genes” (STING) gene, and to single nucleotide polymorphisms (SNPs) in the STING gene, as well as to the STING protein and variants of STING protein resulting from the SNPs.
  • STING Stimulator of Interferon Genes
  • SNPs single nucleotide polymorphisms
  • the invention particularly relates to methods and kits for distinguishing and detecting STING gene SNPs (i.e. STING genotyping) and STING protein variants expressed by humans.
  • the present invention also relates to the uses of the methods and kits as a diagnostic tool to assess the suitability of individuals for STING agonist or antagonist therapy, and also to methods of treatment with a STING agonist or antagonist.
  • STING also known as transmembrane protein 173 is an adaptor protein that binds cyclic dinucleotides (CDN) or agonist compounds, leading to the activation of IKB kinase (IKK) and TANK-binding kinase (TBKi), which when phosphorylated, activate Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFKB) and interferon regulatory factor 3 (IRF3), respectively.
  • INK IKB kinase
  • TKi TANK-binding kinase
  • IRF3 interferon regulatory factor 3
  • Cancer immunotherapy modulates and leverages the host’s immune system to treat cancer, and, over the past decade, there have been significant advances in the field of cancer treatment. Numerous approaches have been explored to elicit or augment anti cancer innate immunity and/or adaptive immunity. Recently, activation of STING, using STING agonists, has shown great potential to enhance antitumor immunity through the induction of a variety of pro-inflammatory cytokines and chemokines, including type I IFNs. A number of natural and synthetic STING agonists have been discovered and developed, and tested in preclinical models and in the clinic for the immunotherapy of various diseases, such as cancer and infectious diseases.
  • cyclic dinucleotides such as cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMP- AMP (cGAMP), are a class of STING agonists that can elicit immune responses.
  • CDNs cyclic dimeric guanosine monophosphate
  • c-di-AMP cyclic dimeric adenosine monophosphate
  • GMP- AMP cyclic GMP- AMP
  • STING antagonists have also become an area of therapeutic interest, as they block overactive STING proteins in various autoimmune diseases. Recently, a number of companies have developed various small molecule STING antagonists, such as C-176 and H-151.
  • SNP single nucleotide polymorphism
  • This HAQ variant has a histidine at the 71 position in place of an arginine, an alanine at the 230 position in place of glycine, and a glutamine at the 293 position in place of arginine.
  • STING variant H232 (R232H) is characterised by a histidine at the 232 position in place of arginine, with an allele frequency of 13.7%.
  • STING haplotype AQ (G230A-R293Q), contains an alanine at the 230 position in place of glycine, and a glutamine at the 293 position in place of arginine, with an allele frequency of 5.2%.
  • STING variant Q contains a glutamine at the 293 position in place of arginine, and has an allele frequency of 1.5% [2].
  • STING protein variants differs in the production of IFNs upon activation.
  • the natural STING ligand cGAMP is a weaker activator of the H232 variant. Therefore, it will be appreciated that the therapeutic application of a STING agonist or an antagonist will be better directed if the identification of the STING alleles present in a specific patient who is about to undergo STING agonist or antagonist treatment is known.
  • a method for determining the genetic polymorphism pattern in the “Stimulator of Interferon Genes” (STING) gene in a subject comprising: amplifying, in a sample obtained from a subject, a sequence of genomic DNA comprising a single nucleotide polymorphism (SNP) within the STING gene; and - performing restriction fragment length polymorphism (RFLP) pattern analysis on the amplified DNA to determine the genetic polymorphism pattern in the STING gene in the sample.
  • SNP single nucleotide polymorphism
  • RFLP restriction fragment length polymorphism
  • an apparatus for determining the genetic polymorphism pattern in the “Stimulator of Interferon Genes” (STING) gene in a subject comprising: means for amplifying, in a sample obtained from a subject, a sequence of genomic DNA comprising a single nucleotide polymorphism (SNP) within the STING gene; and - means for performing restriction fragment length polymorphism (RFLP) pattern analysis on the amplified DNA to determine the genetic polymorphism pattern in the STING gene in the sample.
  • SNP single nucleotide polymorphism
  • RFLP restriction fragment length polymorphism
  • a method for determining the efficacy of a treatment of a subject with a “Stimulator of Interferon Genes” (STING) agonist or a STING antagonist comprising: determining the genetic polymorphism pattern in the Stimulator of Interferon Genes (STING) gene in a sample obtained from a subject using the method according to the first aspect or the apparatus of the second aspect; and determining the suitability of the subject for STING agonist or antagonist therapy based on the genetic polymorphism pattern in the STING gene.
  • STING Stimulator of Interferon Genes
  • a diagnostic or prognostic tool for assessing the suitability of a subject for “Stimulator of Interferon Genes” (STING) agonist or antagonist therapy comprising: - determining the genetic polymorphism pattern in the Stimulator of Interferon
  • STING Genes (STING) gene in a sample obtained from a subject using the method of the first aspect or the apparatus of the second aspect; and determining the suitability of the subject for STING agonist or antagonist therapy based on the genetic polymorphism pattern.
  • a method of treating a subject suffering from a disease characterised by underactive “Stimulator of Interferon Genes” (STING) protein or underexpressed STING gene comprising: amplifying, in a sample obtained from a subject, a sequence of genomic DNA comprising a single nucleotide polymorphism (SNP) within the STING gene; performing restriction fragment length polymorphism (RFLP) pattern analysis on the amplified DNA to determine the genetic polymorphism pattern in the STING gene in the sample; and administering, or having administered, to the subject, a STING agonist, thereby treating the disease.
  • STING Stimulator of Interferon Genes
  • a method of treating a subject suffering from a disease characterised by overactive “Stimulator of Interferon Genes” (STING) protein or overexpressed STING gene comprising: - amplifying, in a sample obtained from a subject, a sequence of genomic DNA comprising a single nucleotide polymorphism (SNP) within the STING gene; performing restriction fragment length polymorphism (RFLP) pattern analysis on the amplified DNA to determine the genetic polymorphism pattern in the STING gene in the sample; and - administering, or having administered, to the subject, a STING antagonist, thereby treating the disease.
  • STING Stimulator of Interferon Genes
  • the methods and apparatus of the first and second aspects of the invention enable the very quick and simple determination of the presence or absence of certain SNPs in the STING gene and, accordingly, the specific STING alleles carried by subjects using tissue or blood samples which can yield very accurate results within only a few hours.
  • the methods and apparatus are inexpensive and have minimal requirements in terms of investment in instrumentation.
  • genotyping can be easily carried out by simple visualisation of restriction fragments by gel electrophoresis, for which no specific software and little technical expertise is required. Considering the number of patients being tested per day per institution, the methods of the invention are both cost-effective and simple to perform. Therefore, the methods provide a very fast and cheap way by which the STING genotype of a patient can be determined.
  • the suitability of a patient for treatment with a STING agonist or antagonist can be readily determined, as per the third and fourth aspects of the invention.
  • the STING gene is located on chromosome 5 at position 31.2, and one embodiment of the coding sequence of STING is known and readily accessible at www.ncbi.nlm.nih.gov.
  • the polymorphism pattern in the STING gene may comprise at least one polymorphism or polymorphic region in the STING gene.
  • the polymorphism pattern in the STING gene comprises more than one, and preferably two, polymorphisms or polymorphic regions in the STING gene.
  • the polymorphism pattern in the STING gene comprises three or more polymorphisms or polymorphic regions in the STING gene.
  • polymorphism can refer to the co-existence, within a population, of more than one form of a gene or portion thereof (e.g. an allelic variant).
  • a portion of a gene of which there are at least two different forms, i.e. two different nucleotide sequences, is referred to as a “polymorphic region of a gene”.
  • a specific genetic sequence at a polymorphic region of a gene is known as an allele.
  • each polymorphic region of the STING gene has at least two different alleles.
  • the sequence variants of each allele may be single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats.
  • a polymorphic region maybe a single nucleotide (i.e. a single nucleotide polymorphism, or SNP), the identity of which differs in different alleles.
  • a polymorphic region can also be several nucleotides long.
  • genotype can refer to the identity of an allele or alleles at one or more polymorphic sites.
  • allelic pattern may consist of either a homozygous or heterozygous state at one or more polymorphic sites.
  • haplotype can refer to a set of genetic or DNA variants, such as SNPs, that are usually inherited together, and these sets of haplotypes are usually located on one chromosome.
  • the alleles making up a haplotype can be located in different places on the chromosome but that are inherited together.
  • the polymorphism pattern in the STING gene may comprise at least one SNP, at least two SNPs, at least three SNPs, or at least four SNPs in the STING gene.
  • a STING variant is R232.
  • an individual has an arginine at position 232. This is the most prevalent human STING variant with an allele frequency of 57.6%, and is therefore considered to be the wild-type.
  • the SNP which encodes R232 is known as rs1131769.
  • the alleles of STING rs1131769 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may comprise detecting or determining the G-allele or the A-allele of the STING rs1131769 SNP.
  • SEQ ID No: 1 The nucleotide sequence encoding one embodiment of human STING variant R232 is referred to herein as SEQ ID No: 1, as follows:
  • the amino acid sequence of one embodiment of human STING variant R232 (bold, underlined) is referred to herein as SEQ ID No: 2, as follows: Therefore, preferably the polymorphism pattern in the STING gene, which is determined, corresponds to STING variant R232.
  • the STING variant R232 comprises or consists of the amino acid sequence substantially as set out in SEQ ID NO:2 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:i.
  • a STING variant is R71H-G230A-R293Q or “HAQ”, in which three SNPs are present.
  • This human STING variant has an allele frequency of 20.4%.
  • a histidine replaces an arginine at position 71; an alanine replaces a glycine at position 230; and a glutamine replaces an arginine at position 293.
  • the SNP which encodes R71H is known as rsii554776.
  • the alleles of STING rsi1554776 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may comprise detecting or determining the G-allele or the A-allele of the STING rsi1554776 SNP.
  • the SNP which encodes G230A is known as rs78233829.
  • the alleles of STING rs78233829 SNP may be identified as (i) a G-allele, and (ii) a C-allele. Therefore, the method may comprise detecting or determining the G-allele or the C-allele of the STING rs78233829 SNP.
  • the SNP which encodes R293Q is known as rs7380824.
  • the alleles of STING rs738o824 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may comprise detecting or determining the G-allele or the A-allele of the STING rs738o824 SNP.
  • SEQ ID No: 3 The nucleotide sequence encoding one embodiment of human STING variant HAQ is referred to herein as SEQ ID No: 3, as follows:
  • amino acid sequence of one embodiment of human STING variant HAQ (bold, underlined) is referred to herein as SEQ ID No: 4, as follows:
  • the polymorphism pattern in the STING gene comprises a SNP which corresponds to STING variant R71H-G230A- R293Q or “HAQ”.
  • the STING variant R71H-G230A-R293Q or “HAQ” comprises or consists of the amino acid sequence substantially as set out in SEQ ID NO:4 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID NO:3-
  • a STING variant is R232H or “H232”, in which one SNP is present.
  • This human STING variant has an allele frequency of 13.7%.
  • a histidine replaces an arginine at position 232.
  • SEQ ID No: 5 The amino acid sequence of one embodiment of human STING variant H232 (bold, underlined) is referred to herein as SEQ ID No: 6, as follows:
  • the polymorphism pattern in the STING gene comprises a SNP which corresponds to STING variant R232H or “H232”.
  • the STING variant R232H or “H232” comprises or consists of the amino acid sequence substantially as set out in SEQ ID No:6 and/ or is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:5.
  • a STING variant is G230A-R293Q or “AQ”, in which two SNPs are present.
  • This human STING variant has an allele frequency of 5.2%.
  • an alanine replaces a glycine at position 230; and a glutamine replaces an arginine a SNP at position 293.
  • the SNP which encodes G230A1S known as rs78233829.
  • the alleles of STING rs78233829 SNP may be identified as (i) a G-allele, and (ii) a C-allele. Therefore, the method may comprise detecting or determining the G-allele or the C-allele of the STING rs78233829 SNP.
  • the SNP which encodes R293Q is known as rs7380824.
  • the alleles of STING rs738o824 SNP maybe identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may comprise detecting or determining the G-allele or the A-allele of the
  • SEQ ID No: 7 The nucleotide sequence encoding one embodiment of human STING variant AQ is referred to herein as SEQ ID No: 7, as follows:
  • the amino acid sequence of one embodiment of human STING variant AQ (bold, underlined) is referred to herein as SEQ ID No: 8, as follows: Therefore, preferably the polymorphism pattern in the STING gene, which is determined, comprises a SNP which corresponds to STING variant G230A-R293Q or “AQ”.
  • the STING variant G230A-R293Q or “AQ” comprises or consists of the amino acid sequence substantially as set out in SEQ ID No:8 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:7.
  • a STING variant is R293Q or “Q”, in which one SNP is present.
  • This human STING variant has an allele frequency of 1.5%.
  • a glutamine replaces an arginine at position 293.
  • the SNP which encodes R293Q is known as rs7380824.
  • the alleles of STING rs738o824 SNP maybe identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may comprise detecting or determining the G-allele or the A-allele of the STING rs738o824 SNP.
  • SEQ ID No: 9 The nucleotide sequence encoding one embodiment of human STING variant Q is referred to herein as SEQ ID No: 9, as follows:
  • amino acid sequence of one embodiment of human STING variant Q (bold, underlined) is referred to herein as SEQ ID No: 10, as follows:
  • the polymorphism pattern in the STING gene comprises a SNP which corresponds to STING variant R293Q or “Q”.
  • the STING variant R293Q or “Q” comprises or consists of the amino acid sequence substantially as set out in SEQ ID No:2 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID No:1.
  • the sample is preferably a biological bodily sample taken from the test subject. Determining the genetic polymorphism pattern in the STING gene in the sample is therefore preferably carried out in vitro.
  • the sample may comprise tissue, blood, plasma, serum, spinal fluid, urine, sweat, saliva, sputum, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof.
  • the sample may be a biopsy.
  • the sample may be contained within the test subject, which maybe an experimental animal (e.g. a mouse or rat) or a human, wherein the method is an in vivo based test.
  • the sample may be an ex vivo sample or an in vitro sample. Therefore, the cells being tested may be in a tissue sample (for ex vivo based tests) or the cells may be grown in culture (an in vitro sample).
  • the biological sample is an ex vivo sample.
  • the sample may be pretreated prior to being used in the invention (e.g. diluted, concentrated, separated, partially purified, frozen etc.).
  • the sample is a pretreated blood sample.
  • the sample may comprise blood, urine, or tissue. Most preferably, therefore, the sample comprises a blood sample.
  • the blood maybe venous or arterial blood.
  • the apparatus may comprise a sample collection container for receiving the extracted sample. Blood samples may be assayed immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the assay is conducted. Detection of SNPs in the STING gene may be carried out on whole blood.
  • the blood sample comprises blood serum.
  • the blood sample comprises nucleated cells.
  • the blood may be further processed before the STING assay is performed, i.e. determining the presence of SNPs in the STING gene.
  • an anticoagulant such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added.
  • the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.
  • the blood sample may be centrifuged or filtered to isolate the nucleated cell fraction of the blood or serum fraction, which may be used for analysis. Hence, it is preferred that the presence of
  • SNPs in the STING gene is analysed or assayed in a nucleated cell fraction of the blood or a blood serum sample. It is preferred that the presence of SNPs in the STING gene is analysed in vitro from a blood serum sample or a nucleated cell fraction of the blood taken from the subject.
  • Amplification techniques for amplifying the sequence of genomic DNA comprising SNPs associated with STING are known to the skilled person and include, but are not limited to, cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (PASA), polymerase chain ligation, nested polymerase chain reaction, and the like.
  • the detecting step may comprise amplification of the sample, for example PCR amplification.
  • PCR involves amplifying DNA, preferably small amounts of DNA, to ease subsequent detection of the genetic polymorphic patterns.
  • PCR-based detection means include multiplex amplification of a plurality of polymorphisms or markers, simultaneously.
  • PCR primers For example, it is well-known to select PCR primers to generate PCR products that do not overlap in size and which can be analysed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labelled and thus can each be differentially detected. Of course, hybridization-based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known to allow multiplex analysis of a plurality of markers.
  • the PCR reaction comprises a mixture of reagents that are well-known in the art in performing the PCR reaction.
  • the mixture may comprise a buffer, forward primer, reverse primer, template, polymerase and water.
  • the PCR may be Quantitative (Q)-PCR, droplet-digital PCR and CrystalTM DigitalTM PCR. All of these techniques are routine in molecular biology and known to those skilled in the art.
  • the activation of DNA polymerase and initial denaturation is carried out at a temperature of between 94 and 98°C, between 95 and 98°C, between 96 and 98°C, or between 97 and 98°C.
  • the initial denaturation is carried out at a temperature of 98°C.
  • the activation of DNA polymerase and initial denaturation is carried out for a period of between 30 seconds and 5 minutes, or between 1 and 4 minutes, or between 2 and 3 minutes.
  • the initial denaturation is carried out for a period of 2 minutes and 45 seconds.
  • PCR is carried out for between 20 and 40 cycles, or between 25 and 35 cycles. Most preferably, PCR is carried out for 30 cycles.
  • the cycle includes the following steps: denaturation; - annealing; and extension.
  • the denaturation step is carried out at a temperature of between 94 and 98°C, between 95 and 98°C, between 96 and 98°C, or between 97 and 98°C. Most preferably, the denaturation step is carried out at a temperature of 98°C.
  • the denaturation step is carried out for a period of between 20 seconds and 2 minutes, between 20 seconds and 1 minute, or between 20 and 30 seconds. Most preferably, the denaturation step is carried out for 30 seconds.
  • the annealing step is carried out at a temperature of between 40 and 60°C, or between 50 and 60°C. Most preferably, the annealing step is carried out at a temperature of 60°C. Preferably, the annealing step is carried out for a period of between 30 seconds and 2 minutes, or between 30 seconds and 1 minute. Most preferably, the annealing step is carried out for 30 seconds.
  • the extension step is carried out at a temperature of between 70 and 75°C, between 71 and 74°C, or between 72 and 73°C. Most preferably, the extension step is carried out at a temperature of 72°C. Preferably, the extension step is carried out for a period of time between 30 seconds and 2 minutes, or between 30 seconds and 1 minute. Most preferably, the extension step is carried out for 30 seconds.
  • PCR includes a final extension step.
  • the final extension step is carried out at a temperature of between 70 and 75°C, between 71 and 74°C, or between 72 and 73°C.
  • the final extension is carried out at a temperature of 72°C.
  • the final extension is carried out for a period of time between 5 and 15 minutes, between 6 and 14 minutes, between 7 and 13 minutes, between 8 and 12 minutes, or between 9 and 11 minutes.
  • the final extension is carried out for a period of 10 minutes.
  • PCR uses a pair of primers, a forward primer and a reverse primer.
  • the forward primer is designed so that it is complementaiy to a sequence of nucleotides upstream of the sequence of interest, whilst the reverse primer is designed so that it is complementary to a sequence of nucleotides downstream of the sequence of interest.
  • the STING nucleotide sequence to which the primer sequences are capable of hybridizing to may be a DNA or RNA sequence.
  • the RNA sequence may be a miRNA, mRNA or siRNA.
  • the term “primer” designates, within the context of the present invention, a nucleotide sequence of that can hybridize specifically to a target genetic sequence and serve to initiate amplification.
  • Primers of the invention may be a single-stranded nucleotide sequence , with a length of between to and 50 nucleotides between 10 and 40 nucleotides, between 10 and 30 nucleotides, between 10 and 25 nucleotides between 11 and 50, between 11 and 40 nucleotides, between 11 and 30 nucleotides, between 11 and 25 nucleotides, between 13 and 50, between 13 and 40 nucleotides, between 13 and 30 nucleotides, between 13 and 25 nucleotides, between 14 and 50, between 14 and 40 nucleotides, between 14 and 30 nucleotides, between 14 and 25 nucleotides, between 15 and 50, between 15 and 40 nucleotides, between 15 and 30 nucleotides, between 15 and 25 nucleotides.
  • primers of the invention have a length of between 15 and 30 nucleotides. More preferably, primers of the invention have a length of between 15 and
  • primers are perfectly matched with the targeted sequence in the STING nucleotide sequence, i.e. having 100% complementarity, allowing specific hybridization thereto and substantially no hybridization to another region.
  • the primers are designed to hybridize to sequences flanking the SNPs which correspond to amino acid residues 71, 230, 232 and 293. This is because substitutions at these residues correspond to the five major STING variants.
  • the forward primer is provided herein as follows:
  • the forward primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 11, or a fragment or variant thereof.
  • the forward primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 11, or a fragment or variant thereof, and amplifies, with its paired reverse primer, the target sequence corresponding to amino acid residue 71.
  • the reverse primer is provided herein as follows: [SEQ ID NO: 12]
  • the reverse primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 12, or a fragment or variant thereof.
  • the reverse primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 12, or a fragment or variant thereof, and amplifies, with its paired forward primer, the target sequence corresponding to amino acid residue 71.
  • the PCR product is 991 bp.
  • the forward primer is provided herein as follows:
  • the forward primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 13, or a fragment or variant thereof.
  • the forward primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 13, or a fragment or variant thereof, and amplifies, with its paired reverse primer, the target sequence corresponding to amino acid residues 230 and 232.
  • the reverse primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 14, or a fragment or variant thereof.
  • the reverse primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 14, or a fragment or variant thereof, and amplifies, with its paired forward primer, the target sequence corresponding to amino acid residues 230 and 232.
  • the PCR product is 501 bp.
  • the forward primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 15, or a fragment or variant thereof.
  • the forward primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 15, or a fragment or variant thereof, and amplifies, with its paired reverse primer, the target sequence corresponding to amino acid residue 293.
  • the reverse primer is provided herein as follows: [SEQ ID NO: 16]
  • the reverse primer comprises or consists of a nucleotide sequence substantially set out as SEQ ID No: 16, or a fragment or variant thereof.
  • the reverse primer is capable of hybridizing to a sequence complementary to the nucleotide sequence as substantially set out in SEQ ID No: 16, or a fragment or variant thereof, and amplifies, with its paired forward primer, the target sequence corresponding to amino acid residue 293.
  • the PCR product is 314 bp.
  • the RFLP pattern analysis comprises subjecting the genomic DNA to restriction enzyme digestion to produce at least one fragment; and subjecting the at least one fragment to gel electrophoresis. The presence of SNPs in the STING gene will result in fragments that display different migration profiles from the wild type fragment patterns.
  • the restriction enzyme digestion reaction comprises a mixture of reagents that are well known in the art in performing the reaction.
  • the mixture may comprise the PCR product, a buffer, a restriction enzyme and water.
  • the restriction enzyme is Hhal.
  • Hhal cuts the genomic DNA at the position corresponding to amino acid residue 71.
  • Hpall may cut the genomic DNA at the position corresponding to amino acid residue 230.
  • the restriction enzyme is EC091I.
  • EC091I cuts the genomic DNA at the position corresponding to amino acid residue 230.
  • the restriction enzyme is Hinill.
  • Hinill cuts the genomic DNA at the position corresponding to amino acid residue 232.
  • the restriction enzyme is Hpall.
  • Hpall cuts the genomic DNA at the position corresponding to amino acid residue 293.
  • the five major STING haplotypes are characterised by the amino acid present at the 71, 230, 232 and 293 positions. Depending on the amino acid present at these positions, and the restriction enzyme used, different length fragments are produced.
  • restriction enzyme digestion produces a 750 bp fragment and a 241 bp fragment. In an embodiment in which histidine is located at the 71 position, restriction enzyme digestion produces a 991 bp fragment.
  • restriction enzyme digestion produces a 317 bp fragment and a 184 bp fragment. In an embodiment in which alanine is present at the 230 position, restriction enzyme digestion produces a 501 bp fragment.
  • restriction enzyme digestion produces a 501 bp fragment.
  • restriction enzyme digestion produces a 317 bp fragment and a 184 bp fragment.
  • restriction enzyme digestion produces a 162 bp fragment, a 116 bp fragment, and a 36 bp fragment.
  • restriction enzyme digestion produces a 198 bp fragment and a 116 bp fragment.
  • the restriction enzyme digestion is carried out at a temperature of between 35 and 40°C, between 36 and 39°C, or between 37 and 38°C. Most preferably, the restriction enzyme digestion is carried out at a temperature of 37°C. Preferably, the restriction enzyme digestion is carried out for a period of between 5 minutes and 4 hours. More preferably, the restriction enzyme digestion is carried out for a period of between 10 minutes and 3 hours, or 15 minutes and 2 hours. Most preferably, the restriction enzyme digestion is carried out for a period of one hour.
  • the fragment is electrophoresed on an agarose gel.
  • concentration of the agarose in the gel is between 0.5% and 4%, or between 1 and 3.5%. Most preferably, the concentration of the agarose in the gel is between 2 and 3%.
  • the fragment is electrophoresed on a polyacrylamide gel.
  • the concentration of the polyacrylamide in the gel is between 5 and 10%. More preferably, the concentration of the polyacrylamide in the gel is between 6 and 9%, or between 7 and 8%. Most preferably, the concentration of the polyacrylamide in the gel is 8%.
  • the polyacrylamide gel is placed in 0.5X TBE buffer.
  • the gel is incubated in ethidium bromide staining solution for between 20 seconds and 5 minutes, or between 30 seconds and 4 minutes, or between 40 seconds and 3 minutes, or between 50 seconds and 2 minutes. Most preferably, the gel is incubated in ethidium bromide staining solution for one minute. Preferably, the gel is visualised using a UV gel documentation system.
  • the method of the fifth aspect may comprise administering, or having administered, to the subject, a STING agonist to treat a disease selected from a group consisting of: cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immune- mediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, mood disorder, sleep disorder, cerebrovascular disease, peripheral artery disease and cardiovascular disease. Most preferably, however, cancer is treated with the STING agonist.
  • STING agonists include synthetic CDN STING agonists and small- molecule STING agonists.
  • CDNs cyclic dinucleotides
  • c-di-GMP cyclic dimeric guanosine monophosphate
  • c-di-AMP cyclic dimeric adenosine monophosphate
  • cGAMP cyclic GMP-AMP
  • STING agonists also include small molecule modulators of STING, of which more information can be found in WO2018/234805, WO2018/234807, WO2019/243825, and WO2019/243823.
  • a STING agonist is administered.
  • an ‘agonist’, an ‘effector’ or an activator, as it relates to STING comprises a molecule, combination of molecules, or a complex, that stimulates STING.
  • the method of the sixth aspect may comprise administering, or having administered, to the subject, a STING antagonist to treat a disease selected from a group consisting of: autoimmune disease, liver fibrosis, fatty liver disease, non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, lupus, sepsis, rheumatoid arthritis (RA), type I diabetes, STING-associated vasculopathy with onset in infancy (SAVI), Aicardi-Goutieres syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus (SLE), retinal vasculopathy, neuroinflammation, systemic inflammatory response syndrome, pancreatitis, cardiovascular disease, non-alcoholic fatty liver disease, renal fibrosis, stroke and age-related macular degeneration (AMD).
  • a disease selected from a group consisting of: autoimmune disease, liver fibrosis, fatty liver disease, non-alcoholic ste
  • STING antagonists include small-molecule STING antagonists, such as C- 176 and H-151, and STING pathway antagonists. Examples of STING antagonists also include small molecule modulators of STING.
  • an ‘antagonist’ as it relates to STING, comprises a molecule, combination of molecules, or a complex, that inhibits, counteracts, downregulates, and/or desensitizes STING.
  • ‘Antagonist’ encompasses any reagent that inhibits a constitutive activity of STING.
  • a constitutive activity is one that is manifest in the absence of a ligand/STING interaction.
  • ‘Antagonist’ also encompasses any reagent that inhibits or prevents a stimulated (or regulated) activity of STING.
  • the “subject” or “individual” maybe a vertebrate, mammal, or domestic animal.
  • medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
  • livestock e.g. a horse
  • pets e.g. a human
  • the subject is a human being.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof.
  • substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-16 and so on.
  • amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the amino acid/polynucleoti de/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate howto calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences.
  • an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g.
  • calculation of percentage identities between two amino acid/polynucleoti de/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs.
  • overhangs are included in the calculation.
  • nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids maybe replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • FIG. 1 is a flow diagram illustrating one embodiment of a STING genotyping method according to the invention
  • FIG. 1 shows SNP typing of human donor Di: STING R232/R232;
  • FIG. 3 shows SNP typing of human donor D2: STING R232/HAQ;
  • Figure 4 shows SNP typing of human donor D3: STING H232/HAQ
  • Figure 5 shows SNP typing of human donor D4: STING R232/H232
  • Figure 6 shows SNP typing of human donor D5: STING HAQ/HAQ;
  • Figure 7 shows SNP typing of human donor D6: STING R232/H232;
  • Figure 8 shows SNP typing of human donor D7: STING H232/HAQ;
  • Figure 9 shows SNP typing of human donor D8: STING R232/R232
  • Figure 10 shows SNP typing of human donor D9: STING R232/R232
  • FIG 11 shows SNP typing of human donor Dio: STING R232/R232;
  • Figure 12 shows SNP typing of human donor D11: STING R232/HAQ; and Figure 13 shows SNP typing of human donor D12: STING R232/R232.
  • the inventors have designed a novel method for rapidly and reliably identifying the presence or absence of mutations in the STING gene and, accordingly, determining which STING alleles a subject carries using tissue or blood samples.
  • the method involves PCR amplification of a SNP-containing sequence of genomic DNA obtained from a human subject. This is followed by RFLP analysis to identify the subject’s STING variant according to the presence or absence of particular SNPs.
  • kits can be used for isolation of genomic DNA as per the manufacturer’s protocol. Such kits are marketed by Qiagen (QIAamp DNA Blood Mini Kit Cat #51104) and Promega (Wizard® Genomic DNA Purification Kit Cat #A1120). In this example, the inventors used the QIAGEN kit. DNA was quantified and measured by monitoring UV absorbance at 260nm using a spectrophotometer (Agilent technologies Cary 60 UV-Vis).
  • PCR for each SNP position PCR was carried out for each SNP position according to the below mentioned PCR conditions and using the primer sets for each SNP amplification set out below.
  • reaction mix was prepared and incubated at 37°C for 1 hour. 5m1 of the digested sample was electrophoresed on 8% polyacrylamide gel in 0.5X TBE buffer for resolution. The gel was incubated in ethidium bromide staining solution for a minute and visualized using a UV gel documentation system.
  • the inventors used their newly designed method to identify the presence or absence of SNPs in the STING gene, and therefore determine the specific STING alleles carried by twelve healthy human donors. They first collected fresh whole blood from each individual healthy donor and then isolated and measured genomic DNA from the samples. PCR amplification of a SNP containing sequence of genomic DNA was then carried out to produce multiple copies of the DNA sample. The inventors then used restriction enzyme digestion to digest the DNA pieces into smaller fragments. Following this, the inventors carried out gel electrophoresis to separate the DNA fragments produced according to their size. Finally, restriction fragment length polymorphism (RFLP) pattern analysis was used to determine the specific STING alleles carried in the genotype of each subject.
  • RFLP restriction fragment length polymorphism
  • the inventors were able to quickly and reliably identify the SNPs present in the STING genes of healthy human donors, and therefore determine the STING variant of the individual.
  • the inventors designed the method to identify SNPs that corresponded to the five major STING variants: R232, HAQ, H232, AQ and Q. Therefore, the PCR primers were designed to hybridize to sequences flanking the SNPs that correspond to amino acid residues 71, 230, 232 and 293, as substitutions at these residues correspond to the five major STING variants.
  • the inventors then used RFLP pattern analysis based on the electrophoretic movement of the DNA fragments to determine the STING variants carried by each individual.
  • human donor Di carries the STING alleles R232/R232. This is because RFLP pattern analysis of the PCR products revealed the presence of arginine at position 71, glycine at position 230, arginine at position 232 and arginine at position 293, based on the fragment lengths. Accordingly, human donor Di carries the STING alleles R232/R232. In comparison, human donor D5 ( Figure 6) carries the STING alleles HAQ/HAQ, since RFLP pattern analysis of PCR products revealed the presence of histidine at position 71, alanine at position 230, arginine at position 232 and glutamine at position 293. By identifying the SNPs and determining the STING variant, this method is extremely informative in revealing that both subjects would be suitable for treatment with a STING agonist.
  • the inventors have developed a novel method for determining the specific STING alleles carried by individuals using tissue or blood samples.
  • this novel method is both rapid and reliable and can therefore yield accurate results within a few hours.
  • this novel method does not require the need for expensive reagents and instruments. Therefore, considering the large number of patients being tested, this method will be both cost effective and feasible to perform. Accordingly, this method can therefore be used as a diagnostic tool to determine the most effective STING agonist therapy for a patient based on their STING genotype.

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Abstract

L'invention concerne un procédé permettant de déterminer le modèle de polymorphisme génétique dans le gène "Stimulateur des gènes de l'interféron" (STING) chez un sujet. Le procédé comprend l'amplification, dans un échantillon prélevé sur un sujet, d'une séquence d'ADN génomique comprenant un polymorphisme nucléotidique unique (SNP) dans le gène STING. Les procédés comprennent également la réalisation d'une analyse du modèle de polymorphisme de longueur des fragments de restriction (RFLP) sur l'ADN amplifié pour déterminer le modèle de polymorphisme génétique dans le gène STING dans l'échantillon. L'invention concerne également des kits permettant de distinguer et de détecter les SNP du gène STING (c'est-à-dire le génotypage STING) et les variants de la protéine STING exprimés chez les êtres humains.
PCT/IB2021/054949 2020-06-08 2021-06-07 Procédé rapide de génotypage de variants sting chez des individus humains WO2021250530A1 (fr)

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