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WO2017055627A1 - Mutations apoc3 pour le diagnostic et la thérapie de la maladie héréditaire amyloïdose rénale - Google Patents

Mutations apoc3 pour le diagnostic et la thérapie de la maladie héréditaire amyloïdose rénale Download PDF

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WO2017055627A1
WO2017055627A1 PCT/EP2016/073570 EP2016073570W WO2017055627A1 WO 2017055627 A1 WO2017055627 A1 WO 2017055627A1 EP 2016073570 W EP2016073570 W EP 2016073570W WO 2017055627 A1 WO2017055627 A1 WO 2017055627A1
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apoc3
iii
apoc
snv
hereditary renal
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PCT/EP2016/073570
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English (en)
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Sophie Valleix
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Descartes
Assistance Publique-Hôpitaux De Paris (Aphp)
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2017055627A1 publication Critical patent/WO2017055627A1/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 invention is in the field of hereditary renal amyloidosis diagnosis and therapy.
  • the invention relates to a specific mutation (or Single Nucleotide Variant, SNV) in human gene APOC3 responsible for hereditary renal amyloidosis.
  • amyloidoses are a group of disorders in which soluble proteins aggregate and deposit extracellularly in tissues as insoluble fibrils, causing progressive organ dysfunction.
  • the kidney is one of the most frequent sites of amyloid deposition in AL, AA, and several of the hereditary amyloidoses.
  • Amyloid fibril formation begins with the misfolding of an amyloidogenic precursor protein.
  • the misfolded variants self-aggregate in a highly ordered manner, generating protofilaments that interact to form fibrils.
  • the fibrils have a characteristic appearance by electron microscopy and generate birefringence under polarized light when stained with Congo red dye. Advances in elucidating the mechanisms of amyloid fibril formation, tissue deposition, and tissue injury have led to identify new target for the diagnosis and therapy for these disorders.
  • apoC-III a small exchangeable apolipoprotein carried in the circulation by VLDL and HDL (1), is a master regulator of plasma TG homeostasis and confers a cardiovasculo-protective effect when its expression is downregulated (2).
  • ApoC-III inhibits the activity of lipoprotein lipase (LPL), the enzyme catalyzing the first and rate-limiting step of TG-rich lipoprotein (TRL) catabolism (3), and also impairs uptake of TRL-remnants, increasing remnant residence time in the circulation (4).
  • LPL lipoprotein lipase
  • TRL TG-rich lipoprotein
  • Heterozygosity for the rare null allele of APOC3, R19X has been associated with high levels of plasma HDL cholesterol (HDL-C), low levels of plasma TG and reduced incidence of ischemic cardiovascular disease (CVD) in the Amish population (5). Subsequently, genome-wide association studies also identified the R19X variant in other population isolates with a strong atheroprotective phenotype (6). More importantly, a recent general population-based study confirmed the association of APOC3 deficiency with an atheroprotective lipid profile and clinical protection against ischemic CVD
  • apoC-III In its lipid bound state, apoC-III is composed of six amphipathic a-helices, the structural motif shared by all exchangeable apo lipoproteins which confers their structural stability (9). Conversely, apolipoproteins in lipid-free form have lower conformational stability and some, such as native and/or variants of apoA-I (10), apoA-II (11), and apoA-IV (12) are prone to self- assembly in vitro and in vivo as amyloid fibrils. Recombinant wild-type apoC-III forms insoluble aggregates in vitro, reminiscent but not typical of amyloid fibrils (13).
  • Amyloid fibril formation is associated with a wide variety of diseases, and the hereditary monogenic forms of human amyloidoses, although rare, provide unique insight into mechanisms of protein misfolding and fibrillogenesis as recently highlighted by the discovery of the amyloidogenic variant of p2-microglobulin (14).
  • a first object of the invention is a method of identifying a subject having or at risk of having or developing hereditary renal amyloidosis, comprising determining, in a sample obtained from said subject, the presence or absence of a single nucleotide variant (SNV) located in APOC3 gene.
  • SNV single nucleotide variant
  • the SNV is ApoC3 : c. l34A>T and wherein :
  • a second object of the invention is a kit for identifying whether a subject has or is at risk of having or developing hereditary renal amyloidosis, comprising:
  • a third object of the invention is a APOC3 antagonist for use in a method of treating hereditary renal amyloidosis and/or preventing progression of hereditary renal amyloidosis in a patient
  • a fourth object of the invention is a nuclease for use in treating hereditary renal amyloidosis and/or preventing progression of hereditary renal amyloidosis in a patient, wherein the presence of SNV in Apoc3 genes in a sample previously obtained from said patient, have been detected by a method of the invention previously described.
  • the term "hereditary renal amyloidosis” or "HRA” is a medical condition which is characterized by progressive renal insufficiency (proteinuric CKD chronic kidney disease) and sometime with sicca syndrome (dryness of the eyes and mouth).
  • the amyloidoses constitute a group of diseases in which proteins deposit extracellularly in tissues as insoluble fibrils. Renal disease is a frequent manifestation of the systemic amyloidoses and often is the major source of morbidity for individuals with these disorders. Without treatment, amyloidosis-associated kidney disease usually progresses to end- stage renal disease (ESRD). To date, approximatively 25 structurally unrelated proteins are known to cause amyloidosis.
  • ESRD end- stage renal disease
  • the initial step in amyloid fibril formation is a misfolding event.
  • the misfolding can result from proteolytic cleavage (e.g., amyloid ⁇ protein), an amino acid substitution (e.g., transthyretin [TTR]), or intrinsic properties that become significant only at high serum concentration or in the presence of specific local factors (e.g., ⁇ 2 -microglobulin).
  • TTR transthyretin
  • the misfolded variants are highly prone to self-aggregation.
  • the self-aggregation generates protofilaments that interact to form fibrils.
  • Amyloid fibrils have a characteristic ⁇ -pleated sheet configuration that produces birefringence under polarized light when stained with Congo red dye.
  • ApoC-III is not known to be an amyloid fibril protein in humans.
  • APOC3 also known as "apolipoprotein C-III” and "APOC-III”;
  • HALP2 means apolipoprotein C-3 (NM 000040 ⁇ NP 000031 apolipoprotein C-III precursor) which is a very low density lipoprotein (VLDL) and high density lipoprotein (HDL) protein.
  • VLDL very low density lipoprotein
  • HDL high density lipoprotein
  • APOC3 inhibits lipoprotein lipase and hepatic lipase; it is thought to delay catabolism of triglyceride - rich particles.
  • the APOA1, APOC3 and APOA4 genes are closely linked in human genomes. An increase in apoC-III levels induces the development of hypertriglyceridemia.
  • the whole sequence of human APOC3 gene is referenced as Gene ID: 345.
  • One example of wild-type APOC3 human amino acid sequence is provided in SEQ ID NO:9 (NCBI Reference Sequence: NP 000031).
  • One example of nucleotide sequence encoding wild-type APOC3 amino acid sequence of SEQ ID NO:9 is provided in SEQ ID NO: 13 (NCBI Reference Sequence: NM 000040).
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the conversion to HRA, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention include time to HRAdisease conversion and therapeutic HRAconversion risk reduction ratios.
  • Risk evaluation or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a HRA condition or to one at risk of developing a HRAdisease.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of HRA diseases, such as amyloid deposit in peripheral tissues, in serum or other fluid (i.e. cerebrospinal fluid), either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to HRA, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a HRA.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for HRA.
  • the present invention may be used so as to help to discriminate those having HRA from normal.
  • “Clinical parameters or indicia” encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), geographical origin (Origin), gender (Sex), family history (FamHX), height (HT), weight (WT), waist (Waist) and body-mass index (BMI), as well as others such as clinical cardinal signs of HRA disease (like renal insufficiency or sicca syndrome).
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or in case of fetus, amniotic fluid or chorionic villy or any other bodily secretion or derivative thereof. In a preferred embodiment, the sample to be tested is saliva or blood. As used herein "blood” includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.
  • the sample is a blood sample or saliva.
  • APOC3 mutations are genomic variants and are detected by using any type of body cell.
  • the cell is a blood cell.
  • the sample comprises APOC3 nucleic acid, wherein APOC3 nucleic acid is genomic DNA.
  • a "subject” in the context of the present invention is preferably a human.
  • Allele has the meaning which is commonly known in the art, that is, an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated results in functional or dysfunctional (including non-existent) gene products.
  • allelic variant means a sequence variation of a gene. Allelic variants can be found in the exons, introns, untranslated regions of the gene, or in the sequences that control expression of the gene. Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype occurs in a sufficiently large population.
  • Single nucleotide variant refers to a type of DNA variation of a single base pair or insertions/deletions. There are millions of SNVs in the human genome. Most commonly, these variations are found in coding sequences of genes, non-coding regions of genes, or in intergenic regions between genes. When SNVs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene's function.
  • SNV pertaining to the invention are unknown (known sequences are publicly available from the data base http://www.ncbi.nlm.nih.gov/SNP/). The mutation studied is described here after:
  • the position of mutation (Asp25Val) on the amino acid sequence of the APOC3 human protein corresponds to APOC3 protein mature form (without signal peptide).
  • the inventors have assayed for a link between a specific variant located at APOC3 gene and hereditary renal amyloidosis (HRA) in HRA patients. More precisely, the inventors have assayed for a link between specific SNV contained in chromosome 11 , especially in APOC3 gene of HRA patients (families).
  • HRA hereditary renal amyloidosis
  • the inventors have performed genetic analysis, histology/immunohistochemistry and proteomic analysis of amyloid deposits in a French kindred with undiagnosed hereditary renal amyloidosis. Screening of DNA blood samples of these undiagnosed HRA family allow to assess the genomic effects of single nucleotide variants (SNVs) in APOC3 gene.
  • SNVs single nucleotide variants
  • SNV biallelic marker located in APOC3 genes wherein the SNV biallelic marker selected from the group consisting of APOC3: c. 134A>T, was not only associated with HRA but cause a new form of hereditary systemic amyloidosis with amyloid deposition in salivary glands, digestive tract, heart, spleen, and vessels.
  • Evidence that this novel form of systemic hereditary amyloidosis is caused by the Asp25 Val mutation in the APOC3 gene in this family have been provided by the genetic study of several members of this French family and the histological/HIC analysis of a lot of affected tissues from several patients carrying the Asp25Val of APOC3.
  • the inventors have then performed functional analysis of recombinant wild-type and variant apoC-III, and demonstrated that the Asp25 Val protein is highly fibrillogenic in its lipid free-state and forms amyloid fibrils under physiological conditions in vitro.
  • APOC3 mutations at exon 3 in particular the Asp25Val mutation of APOC3 which is found in familial HRA case, are strong predictor of HRA disease occurrence in humans.
  • APOC3 mutations at exon3 and reducing expression of the allele carrying this APOC3 amyloidogenic mutation could be a therapeutic target in subject by inhibiting the APOC3 variant expression or activity to delay apoC-III Asp25Val amyloid deposition in tissues.
  • a first object of the invention is a method of identifying a subject having or at risk of having or developing a hereditary renal amyloidosis (HRA), comprising determining, in a sample obtained from said subject, the presence or absence of single nucleotide variant (SNV) located in APOC3 genes.
  • HRA hereditary renal amyloidosis
  • SNV is APOC3: NM_000040:exon3:c. 134A>T and wherein:
  • the method of identifying a subject having or at risk of having or developing a HRAdisease comprising determining the presence or absence of an rare allelic variant located in APOC3genesin a blood, or saliva sample obtained from said subject.
  • said subject may also be one that is asymptomatic for the HRA.
  • an "asymptomatic" subject refers to a subject that does not exhibit HRA symptoms, which are diagnosed, according to internationally validated criteria.
  • said subject may be one that is at risk of having or developing an hereditary renal amyloidosis (HRA), as defined by clinical indicia such as for example: age, gender, clinical marker (like congenital contractures of at least two distinct joints of the body), family history of hereditary renal amyloidosis (HRA).
  • HRA hereditary renal amyloidosis
  • the method of identifying a subject having or at risk of having or developing a hereditary renal amyloidosis comprises a further step by determining the presence or absence of a single nucleotide variant (SNV) located in genes previously known to be responsible for HRA (7TR, FGA, LYZ, GSN, APOA1, APOA2) or new genes (APOC3) described here in a sample obtained from said subject.
  • SNV single nucleotide variant located in genes previously known to be responsible for HRA (7TR, FGA, LYZ, GSN, APOA1, APOA2) or new genes (APOC3) described here in a sample obtained from said subject.
  • the determination of the presence or absence of said SNV may be determined by DNA sequencing, PCR analysis or any genotyping method known in the art.
  • methods include, but are not limited to, chemical assays such as allele specific hybridation, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.
  • the presence or absence of said variant may be detected in a DNA sample, preferably after amplification.
  • the isolated DNA may be subjected to amplification by polymerase chain reaction (PCR), using specific oligonucleotide primers that are specific for the SNV or that enable amplification of a region flanking the SNV.
  • PCR polymerase chain reaction
  • conditions for primer annealing may be chosen to ensure specific amplification; so that the appearance of an amplification product be a diagnostic of the presence of the SNV according to the invention.
  • DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • nucleic acid molecule may be tested for the presence or absence of a restriction site.
  • a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype of the polymorphism.
  • RNA sequencing includes, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase- PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997).
  • DGGE denaturing gradient gel electrophoresis
  • TGGE temperature denaturing gradient gel electrophoresis
  • SSCP single-stranded conformational polymorphism
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; Next-generation sequencing technologies including targeted-NGS panel, exome and whole genome sequencing methods; mass spectrometry sequencing ; sequencing using a chip- based technology; and real-time quantitative PCR.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • OLA may be used for revealing base polymorphisms.
  • two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism.
  • DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.
  • short DNA sequences in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the one of the allele of the polymorphism.
  • Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.
  • the determination of the presence or absence of said SNVs may also be determined by detection or not of the mutated APOC3 protein(s) (i.e. APOC3 Asp25Val protein mature form (without signal peptide)) by any method known in the art.
  • the presence of the protein of interest may be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labelled" with regard to the antibody or aptamer is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5), to the antibody or aptamer, as well as indirect labelling of the probe or antibody (e.g., horseradish peroxidise, HRP) by reactivity with a detectable substance.
  • a detectable substance such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • indocyanine Cy5
  • radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl 11, Rel86 and Rel88.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which may be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, etc.
  • an ELISA method may be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • IHC immunohistochemistry
  • a biological sample or tissue specimen in situ.
  • the overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the target of interest.
  • a biological sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy.
  • Current methods of IHC use either direct labeling or secondary antibody- based or hapten-based labeling.
  • a tissue section e.g. a tissue sample or biopsy
  • a tissue sample or biopsy may be mounted on a slide or other support after incubation with antibodies directed against the APOC- III protein. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed.
  • sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the protein of interest.
  • IHC samples may include, for instance: (a) preparations comprising cell samples (b) fixed and embedded said cells and (c) detecting the protein of interest in said cell samples.
  • an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination. Kit of the invention
  • a second object of the invention is a kit for identifying whether a subject has or is at risk of having or developing an hereditary renal amyloidosis (HRA), comprising:
  • the kit for identifying whether a subject has or is at risk of having or developing a hereditary renal amyloidosis comprising:
  • the kit for identifying whether a subject has or is at risk of having or developing an hereditary renal amyloidosis comprising:
  • the primer or probe may be labeled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.
  • primer sequences for PCR amplification of the APOc3 gene are:
  • APOC3 antagonist inhibitor of expression or of activity
  • a functional assays may be envisaged to determine a APOC3 antagonist, such prevention of amyloid formation, like amyloid formation assays (Amyloid fibrils have a characteristic ⁇ -pleated sheet configuration that produces birefringence under polarized light when stained with Congo red dye) which could be also used to evaluate the ability of APOC3 antagonists to block amyloid formation :
  • fibrate therapy may have therapeutic potential in this new form of amyloidosis. Since the safety and side-effect profiles of fibrates are well known, it might be reasonable to prescribe fibrates to the affected individuals from this kindred in an attempt to slow amyloid formation. This therapy could represent an alternative to the gene silencing approach currently under evaluation in other systemic amyloidosis (34).
  • a third object of the present invention is an APOC3 antagonist for use in treating and/or preventing progression of hereditary renal amyloidosis (HRA) in a patient.
  • a further object of the present invention relates to an APOC3 antagonist which is an inhibitor of the APOC3 expression for use in the treatment of hereditary renal amyloidosis (HRA).
  • HRA hereditary renal amyloidosis
  • RNAs can also function as inhibitors of APOC3 gene expression for use in the present invention.
  • APOC3 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that APOC3 gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • dsRNA targeting APOC3 gene examples include WO2012177947 (Anlylam) and examples of oligonucleotide antisense targeting APOC3 gene are described in WO2012149495(Isis) WO2004097429.
  • Ribozymes can also function as inhibitors of APOC3 gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of APOC3 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of APOC3 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half- life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing APOC3.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of super infection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siR A, shR A or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • Inhibitors of the APOC3 activity is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • a further aspect of the present invention relates to an APOC3 antagonist which is an inhibitor of the APOC3 activity for use in the treatment of hereditary renal amyloidosis (HRA).
  • HRA hereditary renal amyloidosis
  • the present invention relates to compound which is an inhibitor of the APOC3 activity for use in the treatment of hereditary renal amyloidosis (HRA), wherein said compound is an anti-APOC3 antibody which neutralizes APOC3 (see for example Kawakami A, et al., Circulation, 2006: 114: 681-687; KawakamiA et al., Circulation. 2008; 118: 731-742, WO2014131008, WO2004081046, WO2004080375, US2005287137) or an anti- APOC3 antibody fragment which neutralizes APOC3.
  • HRA hereditary renal amyloidosis
  • Antibodies directed against APOC3 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against APOC3 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti-APOC3 single chain antibodies.
  • APOC3 activity inhibitors useful in practicing the present invention also include anti-APOC3 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to APOC3.
  • Humanized anti-APOC3 antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • APOC3 expression inhibitors may be selected from aptamers.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the present invention relates to compound which is an inhibitor of the APOC3 activity for use in the treatment of hereditary renal amyloidosis (HRA), wherein said compound is an PPAR-alpha agonist that significantly inhibit hepatic APOC3 transcription (32,33).
  • HRA hereditary renal amyloidosis
  • PPARa Peroxisome proliferator-activated receptor alpha, also known as NR1C1 (nuclear receptor subfamily 1, group C, member 1)
  • NR1C1 nuclear receptor subfamily 1, group C, member 1
  • fibrate drugs a class of amphipathic carboxylic acids (clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and feno fibrate). They were originally indicated for cholesterol disorders and more recently for disorders that feature high triglycerides.
  • PPAR-alpha agonist have already been largely disclosed in the prior art (see for example Philippe Gervois , Roxane M Mansouri, Expert Opin. Ther. Targets (2012) 16(11): 1113-1125 or Christina Lamerset al Expert Opinion on Therapeutic Patents Jul 2012, Vol. 22, No. 7, Pages 803-841) and one skilled in the art would easily produce them.
  • Another object of the invention is a method for treating a hereditary renal amyloidosis comprising administering to a subject in need thereof a therapeutically effective amount of a APOC3 antagonist as disclosed above.
  • Another object of the invention is a method for treating a hereditary renal amyloidosis comprising administering to a subject in need thereof a therapeutically effective amount of a APOC3 antagonist as disclosed above wherein the presence of SNV at APOC3 gene in a sample previously obtained from said patient, have been detected by a method of the invention previously described.
  • a “therapeutically effective amount” is meant a sufficient amount of compound to treat and/or to prevent the hereditary renal amyloidosis.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the inhibitor of the APOC3 expression or APOC3 activity according to the invention can be administered by any suitable route of administration.
  • the inhibitor according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • the antagonists of the present invention together with one or more conventional adjuvants, carriers, or diluents may be placed into the form of pharmaceutical compositions and unit dosages.
  • the pharmaceutical compositions and unit dosage forms may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredients commensurate with the intended daily dosage range to be employed.
  • compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral uses.
  • Formulations containing about one (1) milligram of active ingredient or, more broadly, about 0.01 to about one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
  • the antagonists of the present invention may be formulated in a wide variety of oral administration dosage forms.
  • the pharmaceutical compositions and dosage forms may comprise compounds of the present invention or pharmaceutically acceptable salts thereof as the active component.
  • the pharmaceutically acceptable carriers may be either solid or liquid. Solid form preparations include powders, tablets, pulls, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier may be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component.
  • the active component In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from about one (1) to about seventy (70) percent of the active compound.
  • Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch gelatin, tragacanth, methylcellulosesodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • preparation is intended to include the formulation of the active compound with an encapsulating material as carrier, providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it.
  • encapsulating material as carrier
  • cachets and lozenges are included. Tablets, powders, capsules, pulls, cachets, and lozenges may be as solid forms suitable for oral administration.
  • liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations.
  • Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia.
  • Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizers, and thickening agents.
  • Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents.
  • Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.
  • the antagonists of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or non-aqueous carriers, diluents solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil, and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • a third object of the present invention is a nuclease for use in treating hereditary renal amyloidosis (HRA) and/or preventing progression of hereditary renal amyloidosis (HRA) in a patient, wherein the presence of SNV at APOC3 gene in a sample previously obtained from said patient, have been detected by a method of the invention previously described.
  • HRA hereditary renal amyloidosis
  • HRA hereditary renal amyloidosis
  • the SNV is selected from the group consisting of APOC3: NM_000040:exon3:c. 134A>T and wherein
  • a man skilled in the art know as to design a specific nuclease in order to repair of genetic point mutations like SNVs located at APOC3 gene, namely APOC3: NM_000040:exon3:c. 134A>T.
  • nuclease or "endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts.
  • the synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALE or CRISPR recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.
  • Restriction endonucleases also called restriction enzymes as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides.
  • some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical.
  • the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and/or reduced catalytic activity when compared to the wildtype restriction endonuclease.
  • the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonucleases subunits.
  • the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites.
  • the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease.
  • the preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides.
  • the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity.
  • the type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of: Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, Bffl, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfrlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pacl, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, andNdel.
  • nuclease for use in the present invention are disclosed in WO 2010/079430, WO2011072246, WO2013045480, Mussolino C, et al (Curr Opin Biotechnol. 2012 Oct;23(5):644-50) and Papaioannou I. et al (Expert Opinion on Biological Therapy, March 2012, Vol. 12, No. 3 : 329-342) all of which are herein incorporated by reference.
  • Another object of the present invention is a method of treating hereditary renal amyloidosis (HRA) in a subject comprising the steps of:
  • step b) detecting in a biological sample obtained at step a) the presence or absence of an allelic variant of a single nucleotide variant (SNV) located inAPOC3 gene; and
  • the SNV is selected from the group consisting of APOC3: NM_000040:exon3:c. 134 A>T and wherein
  • nuclease will allow to modify the nucleotide T in nucleotide A.
  • the invention will be further illustrated by the following figures and examples.
  • FIGURES Figure 1. Apolipoprotein C-III amyloidosis in a French family.
  • Panel A shows family tree with the affected kindred, presenting with systemic amyloidosis and decreased plasma triglyceride levels, both transmitted as autosomal dominant traits.
  • ffi APOC3mutation present; 3 ⁇ 4 clinical syndrome; ff ApoC-III amyloid confirmed histologically;FUDecreased plasma triglyceride levels; Hno clinical syndrome, no amyloid on histology, absence of APOC3 mutation, no hypotriglyceridemia.
  • Panel B shows abundant vascular, and moderate glomerular and interstitial amyloid deposits in a kidney specimen from the proband III.3 (top-left panel: Congo-red stained section viewed under polarized light showing bright birefringence confirming amyloid deposition, original magnification x200; bottom-left panel: immunofluorescence with anti-apoC-III antibody co-localizing with amyloid; original magnification x 200); top-right panel: electron micrograph of amyloid fibrils on immunogold staining with anti-apoC-III antibody, original magnification x 80000); bottom- right panel: Amyloid deposits are seen in both the mesangium and the glomerular arteriole leading to a slight ischemic feature of glomerular capillaries, probably explaining the renovascular HTA (Jones staining, X200).
  • Panel C shows proteomic analysis of amyloid plaques from the salivary gland biopsy specimen of patient IV.3. The 30 most abundant proteins identified are listed and data indicate the presence of the apoC-III protein carrying the mutated amino acid along with the apolipoprotein E and the serum amyloid P component.
  • Panel D shows apoC-III tryptic peptide coverage: the top line represents the sequence of wild type apoC- III sequence with the 20 amino acid signal peptide (WT) (SEQ ID N°9); the peptides identified from the amyloid plaques (p.Asp45Val or Asp25Val) are indicated by the red (peptides outside the site of the mutation) and green (the peptide (SEQ ID N°10) corresponding to the site of the mutation with valine residue instead of aspartate) squares.
  • WT 20 amino acid signal peptide
  • Panel E shows partial sequence chromatograms of exon 3 of the APOC3 gene (SEQ ID N°l l and SEQ ID N°12) with the heterozygous nucleotide substitution (c.l34A>T: Asp25Val or p.Asp45Val) identified in the six affected patients (family members II.3, III.3, III.5, IV.1, IV.3, and IV.4), and was absent in the two healthy individuals (III.1 , IV.2) where no amyloid deposits were found in their salivary gland biopsies.
  • Panel A represents mass spectrometry analysis of apoC-III isoforms from HDL2 and
  • apoC-IIIO Asp25Val-carriers
  • apoC-IIIl apoC-IIIl
  • apoC-1112 where the index represents the number of sialic acid residues respectively.
  • the subscript lower case letter indicates the absence of glycans (apoC-IIIOa) or the presence of a GalNAc-Gal disaccharide (apoC-IIIOc) respectively.
  • the arrow points to the peak corresponding to the Asp25Val variant of apoC-III isoforms with a reduced mass of 15.5 Da (apoC-IIIl : 9406.11; apoC-1112: 9708.90) in comparison to the wild type apoC-III isoforms (apoC-IIIl : 9421.61; apoC-1112: 9714.40).
  • the variant apoC-IIIl and apoC-1112 isoforms are detected only in Asp25Val-carriers.
  • Panel B shows the relative quantification of each apoC-III isoform in three Asp25Val-carriers (III.3, IV.3, and IV.4) in comparison with a control subject (III.4).
  • the ratio of apoC-III isoforms is modified in the Asp25Val-carriers and less Asp25Val variant is present in HDL2 and HDL3 than wild type apoC-III.
  • Panel C shows FPLC size exclusion chromatogram of lipoproteins highlighting the drastic decrease of VLDL and the massive increase of HDL particles in the proband III.3 in comparison to a control subject.
  • Panel D shows cholesterol efflux capacity of small, dense HDL3c and total HDL from the proband (III.3) and one healthy normolipidemic control subject compared on the basis of unit PL mass content in THP-1 cells, expressed as % [3H]-cholesterol efflux.
  • Panel E represents the antioxidative activity of small, dense HDL3c and total HDL from the proband (III.3) and one healthy normolipidemic control subject towards LDL oxidation, expressed as an accumulation of lipid hydroperoxides with conjugated diene structure.
  • Panel A shows the ⁇ -sheet content predicted with the sequence-based Chou-Fasman algorithm.
  • Panel B shows the aggregation propensity calculated according to the Zyaggregator method.
  • Panel C shows the change in secondary structure monitored by far-UV CD during aggregation of both wild type (WT) and D25V ApoC-III in PBS pH 7.4, 37°C and 1,500 rpm. CD spectra of samples (200 ⁇ ) at 0, 0.5, 4, 6 h hours respectively were recorded in 1.0 mm path length quartz cell, buffer subtracted and expressed as mean ellipticity per residue ( ).
  • Panel D shows the increase in turbidity at 350 nm which describes the aggregation of WT and D25V ApoC-III.
  • An asterisk (*) highlights the position of residue 25.
  • Panel B shows amide chemical shift differences projected onto the WT NMR structure (pdb2jq3) according to the indicated color scale. Grey areas indicate unassigned residues or proline.
  • Panel C shows the TRACT relaxation interference measurements35 of WT and D25V apoC-III, determined by integration of the amide envelope and fitting to single exponential decays.
  • Fitted 15N relaxation rates were 7.2 ⁇ 0.4 s-1 (WT) and 7.2 ⁇ 0.2 s-1 (D25V) in the a spin state, and 19.3 ⁇ 1.0 s-1 (WT) and 18.2 ⁇ 0.4 s-1 (D25V) in the ⁇ spin state, corresponding to hydrodynamic radii of 20.3 ⁇ 0.6 A (WT) and 19.6 ⁇ 0.2 A (D25V).
  • Genomic DNAs were extracted from peripheral blood samples and the promoter region, coding regions of APOC3 were amplified by PCR and sequenced. Mutation nomenclature was based on the APOC3 transcript reference (NCBI RefSeqcDNA accession number NM 004048), and according to Human Genome Variation Society guidelines. Therefore, the apoC-III variant is described as p.Asp45Val (with the signal peptide) whereas the mature protein is designed as Asp25Val.
  • Amyloid was detected by Congo red staining of formalin fixed wax-embedded biopsy sections (kidney, salivary glands, heart, bowel, and bronchia from III.3; kidney, salivary glands, skin, heart, liver from III.5; salivary glands, kidney, skin, bowel from II.3; salivary glands from IV.1, IV.3, and IV.4).
  • 3 ⁇ cryostat sections were stained using rabbit polyclonal fluorescein isothiocyanate (FITC) conjugates specific for serum anti- apoC-III antibody (Abnova, Taiwan), and FITC-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Specificity of staining was confirmed, and positive and negative controls were included in each case.
  • Gold-conjugated goat anti-rabbit IgG was used for immuno-electron microscopy and ultra-thin sections were processed for electron microscopy studies (14).
  • Amyloid deposits were microdissected from salivary glands (IV.3 and IV.4) and heart
  • Plasma levels of triglycerides, apoC-III concentrations, and HDL cholesterol were measured in the non-fasting state using standard biochemical assays.
  • Lipoproteins of III.3 and of a control were analysed by Fast Protein Liquid Chromatography from 200 ⁇ serum using two Superose 6 columns connected in series. The elution rate was 0.4 ml/min; 200 ⁇ fractions were collected and TG and total cholesterol contents were determined in each tube.
  • Lipoprotein classes were prepared by sequential centrifugation to characterize their lipid and apolipoprotein composition (16). Lipoproteins were also sub fractionated by single step, isopycnic non denaturing density gradient ultracentrifugation and their chemical composition and biological activities were determined (17). The phospholipidome of HDL, their cholesterol efflux capacity and antioxidative activity are reported in Valleix et ah, 2016 (36).
  • Beta Aggregation Propensity Sequence-based Chou-Fasman algorithm was used to predict the beta propensity at the mutation site.
  • the aggregation propensities of wild type and Asp25 Val ApoC-III were predicted with the Zyggregator method.20
  • ApoC-III was expressed from a pET23b vector (36) containing the full length cDNA for human ApoC-III, including the sequence encoding a C-terminal His6-tag preceded by two additional residues (Leu and Glu) (21).
  • Fibrillogenesis experiments were performed in standard quartz cells stirred at 1 ,500 rpm at 37°C using 100 ⁇ ApoC-III isoforms in phosphate buffered saline, pH 7.4 (PBS). Aggregation was carried out without seeds of preformed fibrils and the increase in turbidity at 350 nm was monitored. Atomic force microscopy (AFM) analysis was carried out on 10 ⁇ of fibrillar sample incubated on a freshly cleaved mica substrate for 5 min, then rinsed and dried. Conformational modifications during aggregation of apoC-III were monitored by far-UV CD.
  • AFM Atomic force microscopy
  • NMR spectra were acquired for 0.5 mM samples of wild type and Asp25Val apoC-III (10 mM sodium acetate (pH 5.0), 180 mM SDS, 10% D20) at 315 °K, using a 700 MHz BrukerAvance III spectrometer equipped with a TXI cryoprobe, and processed with nmrPipe and CCPN Analysis.
  • the proband (III.3) (Fig. 1A) presented at age 51 years with sicca syndrome and hypertension, and two years later was found to have CKD with modest proteinuria (0.4 g/L).
  • Salivary gland and renal biopsies showed amyloid deposits (Fig. IB).
  • a search for an underlying monoclonal gammopathy was negative, and plasma concentrations of serum amyloid A protein (SAA) and C-reactive protein (CRP) were in the normal range.
  • SAA serum amyloid A protein
  • CRP C-reactive protein
  • the proband reached end-stage renal disease (ESRD) aged 56 years and received acadaveric kidney transplant uneventfully.
  • Amyloid deposits were discovered in the salivary glands of six affected patients as well as in a variety of other tissues (see Methods). Renal histology showed abundant amyloid deposits in the walls of renal arterioles, and only moderate amyloid in the glomeruli consistent with the modest proteinuria (Fig. IB). Immunohistochemical staining and proteomic methodologies were combined in order to characterize the amyloid fibril protein from different pathological tissues in five affected family members (III.3, III.5, IV.1, IV.3, and IV.4). In all cases, antibodies to apoC III bound specifically to the amyloid deposits (Fig. IB). Proteomic analysis of amyloid deposits from the heart of patient III.3 and salivary glands of individuals IV.3 and IV.4 confirmed that the Asp25Val apoC-III variant was the fibril protein (Fig. 1C and ID).
  • Asp25Val-carriers displayed a 30 to 50% decrease in plasma apoC III concentration, hypotriglyceridemia and increased plasma levels of HDL-C as compared with non-carriers in the family (Table 1A).
  • AP0C3 promoter polymorphism genotypes were determined among family members and no correlation with plasma triglyceride levels was found (Fig.
  • the profile of multiple lipid classes in the proband's HDL was altered (Table ID). Although the proband had CKD, the intrinsic cholesterol efflux capacity of his HDL was elevated compared to the control, and the antioxidative activities of total HDL and small, dense HDL3c were similar between proband and control subjects (Fig. 2E).
  • the phenotype of this form of amyloidosis is characterized by onset with sicca syndrome and progressive renal insufficiency leading to ESRD.ApoC-III should be added to the list of protein variants associated with hereditary renal amyloidosis (22), along with apoA-I (10), apoA-11,11 lysozyme (23), fibrinogen Aa-chain (24), and gelsolin (25).
  • the diagnosis of apoC-III amyloidosis may be suggested by hypotriglyceridemia, a constant biological marker that preceded onset of clinical symptoms in amyloidotic patients from this kindred.
  • Asp25Val-carriers displayed hypotriglyceridemia associated with a dramatic decrease in the number of VLDL particles and a concomitant massive increase in the larger HDL2 fraction, most probably due to accelerated VLDL lipolysis. Indeed, it is well known that surface lipids of VLDL which are liberated after VLDL-TG hydrolysis by LPL are transferred to HDL by phospholipid transfer protein (PLTP), resulting in an increase in HDL size and particle number as confirmed in mice deficient in LPL (27) and PLTP (28) which lack HDL.
  • PLTP phospholipid transfer protein
  • fibrate therapy may have therapeutic potential in this new form of amyloidosis. Since the safety and side-effect profiles of fibrates are well known, it might be reasonable to prescribe fibrates to the affected individuals from this kindred in an attempt to slow amyloid formation. If successful, this therapy could represent an alternative to the gene silencing approach currently under evaluation in other systemic amyloidosis (34).
  • the proband corresponds to individual III 3
  • Proband 716 0.59 18.1 ⁇ .211 1.2 1.1 0.030 0.008 0.013

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Abstract

La présente invention concerne un procédé d'identification d'un sujet ayant ou présentant un risque d'avoir ou de développer une amyloïdose rénale héréditaire, comprenant la détermination, dans un prélèvement obtenu auprès dudit sujet, de la présence ou de l'absence d'un variant nucléotidique unique (SNV) du gène APOC3, tel que APOC3: c.134>T qui conduit à la substitution de l'acide aminé D25V dans la protéine APOC3.
PCT/EP2016/073570 2015-10-02 2016-10-03 Mutations apoc3 pour le diagnostic et la thérapie de la maladie héréditaire amyloïdose rénale WO2017055627A1 (fr)

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WO2020135673A1 (fr) * 2018-12-28 2020-07-02 苏州瑞博生物技术有限公司 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés
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US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
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US11896674B2 (en) 2018-09-30 2024-02-13 Suzhou Ribo Life Science Co., Ltd. SiRNA conjugate, preparation method therefor and use thereof
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US12084661B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US12274752B2 (en) 2017-12-01 2025-04-15 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof

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US11091539B2 (en) 2016-07-08 2021-08-17 Staten Biotechnology B.V. Anti-ApoC3 antibodies and methods of use thereof
US11242381B2 (en) 2017-04-21 2022-02-08 Staten Biotechnology B.V. Anti-ApoC3 antibodies and methods of use thereof
US10538583B2 (en) 2017-10-31 2020-01-21 Staten Biotechnology B.V. Anti-APOC3 antibodies and compositions thereof
US11248041B2 (en) 2017-10-31 2022-02-15 Staten Biotechnology B.V. Anti-ApoC3 antibodies
US11248042B2 (en) 2017-10-31 2022-02-15 Staten Biotechnology B.V. Polynucleotides encoding anti-ApoC3 antibodies
US11492620B2 (en) 2017-12-01 2022-11-08 Suzhou Ribo Life Science Co., Ltd. Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof
US11660347B2 (en) 2017-12-01 2023-05-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
US12083142B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US12084661B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US12274752B2 (en) 2017-12-01 2025-04-15 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
CN108103181A (zh) * 2017-12-15 2018-06-01 苏州国科闻普生物科技有限公司 高甘油三酯血症突变位点的检测方法和检测试剂盒
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
US11918600B2 (en) 2018-08-21 2024-03-05 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof
US11896674B2 (en) 2018-09-30 2024-02-13 Suzhou Ribo Life Science Co., Ltd. SiRNA conjugate, preparation method therefor and use thereof
CN112423794A (zh) * 2018-12-28 2021-02-26 苏州瑞博生物技术股份有限公司 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途
WO2020135673A1 (fr) * 2018-12-28 2020-07-02 苏州瑞博生物技术有限公司 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés

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