WO2017055627A1 - Apoc3 mutations for the diagnosis and therapy of hereditary renal amyloidosis disease - Google Patents

Abstract

The present invention relates to a method of identifying a subject having or at risk of having or developing a hereditary renal amyloidosis, comprising determining, in a sample obtained from said subject, the presence or absence of a single nucleotide variant (SNV) in APOC3 gene, such as APOC3: c.134>T which leads to the D25V amino acid substitution in the APOC3 protein.

Description

APOC3 MUTATIONS FOR THE DIAGNOSIS AND THERAPY OF

HEREDITARY RENAL AMYLOIDOSIS DISEASE

FIELD OF THE INVENTION:

The invention is in the field of hereditary renal amyloidosis diagnosis and therapy. In particular, the invention relates to a specific mutation (or Single Nucleotide Variant, SNV) in human gene APOC3 responsible for hereditary renal amyloidosis.

BACKGROUND OF THE INVENTION:

The 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.

There is growing evidence that 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). 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

(7,8). 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).

Here inventors report a French family with severe renal amyloidosis and hypotriglyceridemia, both caused by a novel Asp25Val-apoC-III variant. Remarkably, this variant induced a favourable, as yet undescribed, lipoprotein profile, despite chronic kidney disease (CKD). The Asp25Val protein is highly fibrillogenic in its lipid free-state and forms amyloid fibrils under physiological conditions in vitro. This study provides insights into the genetics of apoC-III and HDL metabolism in health and disease.

SUMMARY OF THE INVENTION:

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.

In a preferred embodiment, the SNV is ApoC3 : c. l34A>T and wherein :

- the presence of the allele (T) of ApoC3::c. l34A>T indicates an increased risk of having or developing hereditary renal amyloidosis;

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:

- Means for detecting the SNV selected from the group consisting of ApoC3::c.l34A>T and

- instructions for use. 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. DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

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. For each of these amyloidogenic "precursor proteins," the initial step in amyloid fibril formation is a misfolding event. The misfolding can result from proteolytic cleavage (e.g., amyloid β 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., β2 -microglobulin). Regardless of the protein or the trigger for misfolding, the misfolded variants are highly prone to self-aggregation. The self-aggregation generates protofilaments that interact to form fibrils. Amyloid fibrils have a characteristic β-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.

Patients carrying the c. l34A>T (Asp25Val) deleterious mutation in APOC3 gene develop a HRA with a severe renal insufficiency (CKD) and hypotriglyceridemia. As determined by study of the APOC3 mutant, concordance between the APOC3 mutation and presence of amyloid among patients, coupled with identification of Asp25Val apoC-III within amyloid fibrils and in vitro demonstration of its amyloidogenicity, establishes that the Asp25Val variant causes the amyloidosis. Therefore, mutation of APOC3 genes is responsible for novel cause of systemic hereditary renal amyloidosis. The term "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. 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. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for HRA. In other embodiments, 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).

A "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.

In a preferred embodiment the sample is a blood sample or saliva.

According to the invention, APOC3 mutations are genomic variants and are detected by using any type of body cell. In a preferred embodiment the cell is a blood cell.

According to the invention, 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.

The term "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.

The term "mutation" or "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.

The term "Single nucleotide variant" or "SNV" 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.

The 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).

Mutation nomenclature was based on the APOC3 transcript reference (NCBI

RefSeqcDNA accession number NM 000040.1), and nucleotides were numbered according to the cDNA with +1 corresponding to the A of ATG translation initiation codon according to Human Genome Variation Society guidelines, http://www.hgvs.Org/mutnom//. Therefore, the apoC-III variant is described as p.Asp45Val (with the signal peptide) whereas the mature protein is designed as D25V.

Diagnostic method:

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).

As disclosed in the examples herein, 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.

More precisely, the inventors have now identified specific 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.

These results support that the 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. Furthermore, 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.

In particular embodimentthe SNV is APOC3: NM_000040:exon3:c. 134A>T and wherein:

- the presence of the allele (T) of APOC3: NM_000040:exon3:c. 134A>T indicates a highrisk of having or developing hereditary renal amyloidosis (HRA). In one embodiment of the invention, 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. In another embodiment, said subject may also be one that is asymptomatic for the HRA. As used herein, an "asymptomatic" subject refers to a subject that does not exhibit HRA symptoms, which are diagnosed, according to internationally validated criteria.

In another embodiment of the invention, 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).

Accordingly, in one embodiment of the invention, the method of identifying a subject having or at risk of having or developing a hereditary renal amyloidosis (HRA) as previously described, 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.

According to the invention, 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. Examples of such 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.

For example, the presence or absence of said variant may be detected in a DNA sample, preferably after amplification. For instance, 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. According to a first alternative, 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. Otherwise, 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.

Actually numerous strategies for genotype analysis are available (Cooper et al, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype of the polymorphism. Further strategies include, 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). 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. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, 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.

Therefore, 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.

According to the invention, 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. As used herein, 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. An antibody or aptamer may be also labelled with a radioactive molecule by any method known in the art. For example, 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.

More particularly, 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.

Alternatively, an immunohistochemistry (IHC) method may be used. IHC specifically provides a method of detecting a target in 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. Typically 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. Examples of known IHC systems include, for example, En Vision™ (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBA™ kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine® (Nichirei Corp, Tokyo, Japan). In particular embodiment, a tissue section (e.g. 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. For the production of photomicrographs, 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. Therefore 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. In some embodiments, 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:

- means for detecting the SNV located in APOC3 gene

and

- instructions for use.

In one embodiment of the invention the kit for identifying whether a subject has or is at risk of having or developing a hereditary renal amyloidosis (HRA), comprising:

- means for detecting the SNV selected from the group consisting of APOC3: NM_000040:exon3:c. 134A>T and

- instructions for use.

In one embodiment of the invention, the kit for identifying whether a subject has or is at risk of having or developing an hereditary renal amyloidosis (HRA), comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a SNV consisting of APOC3: NM_000040:exon3:c. 134A>T and

- instructions for use.

In one embodiment of the invention, 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. Example of primer sequences for PCR amplification of the APOc3 gene are:

Exon 1 APOC3_ IF caagccacccacttgttctc (SEQ ID N°l)

APOC3 JR tccgaggcttccttagctc (SEQ ID N°2)

Exon 2 APOC3_ 2F cttctggcagacccagctaa (SEQ ID N°3)

APOC3 _2R gaccacccattgggactg (SEQ ID N°4)

Exon 3 APOC3 _3F atgggtggtcaagcagga (SEQ ID N°5)

APOC3 _3R gagcacctccattccattgt (SEQ ID N°6)

Exon 4 APOC3 _4F ctgactggtgtcgtccagtg (SEQ ID N°7)

APOC3 _4R ccctggagattgcaggac (SEQ ID N°8)

Therapeutic method As previously mentioned the inventors demonstrate in the functional analysis of APOC3 mutant in vitro model, highly fibrillogenic in its lipid free-state and forms amyloid fibrils under physiological conditions similar to those observed in HRA patients.

APOC3 antagonist

Reducing the amyloid fibril precursor protein concentration is known to slow amyloid formation and improve prognosis among patients with a variety of systemic amyloidosis. Accordingly use of APOC3 antagonist (inhibitor of expression or of activity) may be used to treat this new form of amyloidosis.

Accordingly the present invention, 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 β-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 :

Furthermore since PPARa agonists (fibrates) significantly inhibit hepatic APOC3 transcription (32,33), 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).

Accordingly 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. Inhibitors of the APOC3 expression

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).

Small inhibitory R As (siRNAs) 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). 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. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Examples of dsRNA targeting APOC3 gene are described in 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. Specific 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.

Both 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. In its broadest sense, 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. Preferably, 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. In general, 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. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. 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. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which 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. Reportedly, 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. In addition, wild- type 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.

Other vectors include plasmid vectors. 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. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, 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. It may also be administered into the epidermis or a mucosal surface using a gene-gun. 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. In a preferred embodiment, 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

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).

In a particular embodiment, 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.

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. Various adjuvants known in the art can be used to enhance antibody production. Although 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). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) 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. Alternatively, 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. For the most part, 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. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of APOC3 are selected.

In still another embodiment, 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. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. 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).

Then, for this invention, neutralizing aptamers of APOC3 are selected. In a particular embodiment, 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). "PPARa" (Peroxisome proliferator-activated receptor alpha, also known as NR1C1 (nuclear receptor subfamily 1, group C, member 1)) is the main target of 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.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat and/or to prevent the hereditary renal amyloidosis.

It will be understood that 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. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The inhibitor of the APOC3 expression or APOC3 activity according to the invention can be administered by any suitable route of administration. For example, 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. The pharmaceutical 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. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided 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.

The term "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. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pulls, cachets, and lozenges may be as solid forms suitable for oral administration.

Other forms suitable for oral administration include 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. Examples of oily or non-aqueous carriers, diluents solvents or vehicles 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. Alternatively, 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. Nuclease Therapy Targeting Apoc3

Accordingly 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.

In particular embodiment the SNV is selected from the group consisting of APOC3: NM_000040:exon3:c. 134A>T and wherein

- the presence of the allele (T) of APOC3: NM_00002:exon3:c. 134A>T is indicative of hereditary renal amyloidosis (HRA).

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.

The term "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. In contrast, 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. Moreover, also preferably 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.

According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonucleases subunits. Preferably, in accordance with the present invention 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. Preferably, 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. Preferably, 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.

Other 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:

a) providing a biological sample from a subject,

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

if a SNVs is detected,

treating the subject with an nuclease to modify said SNV and restoring the wild type sequence.

In particular embodiment the SNV is selected from the group consisting of APOC3: NM_000040:exon3:c. 134 A>T and wherein

- the presence of the allele (T) of APOC3: NM_000040:exon3:c. 134A>T is indicative of hereditary renal amyloidosis (HRA).

Accordingly, the nuclease will allow to modify the nucleotide T in nucleotide A. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

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; ¾ 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). Parallel control negative sections consisting of the absence of the primary apoC-III antibody are seen in lower magnifications, demonstrating the specificity of the apoC-III antibody. 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. These results clearly show that amyloid plaques contained only the full-length mutated apoC-III protein but not the wild type protein. 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. D25V+/-, heterozygous for the apoC-III amyloidogenic variant; D25V-/-, absence of apoC-III mutation.APOC3 haplotypes distribution of C-482T and T-455C polymorphism is shown, and indicates lack of association between APOC3 promoter polymorphisms and plasma TG levels.

Figure 2. Lipoprotein studies, characterization and quantification of wild type and Asp25Val apoC-III isoforms.

Panel A represents mass spectrometry analysis of apoC-III isoforms from HDL2 and

HDL3 of Asp25Val-carriers (Asp25Val +/-) in comparison with a non-carrier of the variant (Asp25Val -/-). Three apoC-III isoforms are commonly referred to as apoC-IIIO, apoC-IIIl, and 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.

Figure 3. Prediction and experimental evidence of the beta aggregation propensity at the site of mutation.

Panel A shows the β-sheet content predicted with the sequence-based Chou-Fasman algorithm. Panel B shows the aggregation propensity calculated according to the Zyaggregator method.²⁰ 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 μΐ) 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.

Figure 4. NMR spectroscopy.

Panel A shows amide chemical shift differences between WT and D25V apoC-III, calculated as the weighted combination ΔδΝΗ = (Δ5Η2+ (ΔδΝ/ 5)2)1/2. 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 β spin state, corresponding to hydrodynamic radii of 20.3 ± 0.6 A (WT) and 19.6 ± 0.2 A (D25V). EXAMPLE:

Materiel & Methods

The study was carried out in accordance with the Declaration of Helsinki. All authors vouch for the completeness and accuracy of the analyses and results. All patients provided written informed consent.

Genetic Analysis

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.

Histology And Immunohistochemistry

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). For indirect immunofluorescence, 3 μιη 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).

Laser Microdissection and Tandem Mass Spectrometry-Based Proteomics

Amyloid deposits were microdissected from salivary glands (IV.3 and IV.4) and heart

(III.3) and analyzed by tandem mass spectrometry, as previously described (15). Algorithms to identify the mutant tryptic apoC-III peptide were updated within Swissprot, and a second-round data analysis was performed.

Lipids and Lipoproteins Analyses

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 μΐ serum using two Superose 6 columns connected in series. The elution rate was 0.4 ml/min; 200 μΐ 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).

Mass Spectrometry of APOC-III isoforms from HDL

20 μg of HDL fraction was dissolved and acidified in 20 μΐ of 0.1 % TFA (trifluoroacetic acid) for 15 min with agitation. The protein was desalted using ZipTips C18 (Millipore) following the manufacturer instructions and eluted with 20 μΐ of saturated matrix solution of sinapinic acid in 50% acetonitrile and 0.5% (v/v) TFA. The mass spectrometric measurements were performed using a MALDI TOF/TOF 4800 Proteomics Analyzer mass spectrometer, as previously described (18). The MS spectra from m/z 8000-10,000 Da were acquired both in reflectron and in linear positive ion modes using 1000 laser shots.

Radiolabeled Sap Scintigraphy

Patients III.3, IV.3, and IV.4 underwent 1231-labeled serum amyloid P component

(SAP) scintigraphy.19

Prediction Of The 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

Expression And Purification Of Recombinant Wild Type And Variant Asp25val APOC-III

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

Fibrillogenesis experiments were performed in standard quartz cells stirred at 1 ,500 rpm at 37°C using 100 μΜ 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 μΐ 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.

NMR Spectroscopy

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.

Results

Patients

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. The proband reached end-stage renal disease (ESRD) aged 56 years and received acadaveric kidney transplant uneventfully. 1231 SAP scintigraphy, undertaken just before renal transplantation, showed a small total body amyloid load in the spleen and kidneys, at which point a fat aspirate demonstrated extensive amyloid (data not shown (36)). There was a strong family history of amyloidosis, (Fig. 1A) with all affected members suffering from sicca syndrome and mildly proteinuric CKD.

Histology

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).

APOC3 genotype, plasma triglyceride and APOC-III levels

Five available affected patients were heterozygous for a single base substitution

(c. l34A>T) in exon 3 of APOC3, encoding replacement of the negatively charged aspartate residue at position 25 of the mature apoC-III by a valine residue (Asp25Val) (Fig. IE). No amyloidogenic mutations were identified in the genes for any other known amyloid fibril proteins. Moreover, APOC3 sequences of two unaffected family members (III.l and IV.2) with no amyloid deposits on salivary gland biopsy were wild-type.

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. lA).We characterized by MS apoC-III isoforms in HDL2/HDL3 of hypotriglyceridemic Asp25Val- carriers and normotriglyceridemic non Asp25Val-carriers.We found higher levels of di- sialylated apoC-III isoform in Asp25 Val-carriers compared with controls, with a corresponding decrease of mono- and non-sialylated apoC-III isoforms (Fig. 2A and 2B).MS analysis further showed that the Asp25Val-variant represented no more than 30% of total HDL apoC-III (Fig. 2A and 2B).

Lipoprotein Studies

The lipoprotein profiles of III.3 and a normolipidemic subject were substantially different. Interestingly, VLDL was nearly absent, LDL was reduced, and HDL was massively increased in the proband's plasma (Fig. 2C). A detailed analysis of all lipoprotein constituents is shown in Table IB. There was a 5 -fold increase in HDL2 but lower apoC-III content in the proband's HDL2/HDL3 fractions (Data not shown (36)). Additionally, HDL particles from III.3 were enriched in esterified and free cholesterol, and in phospholipid, but depleted in TG and protein, a composition typical of hyperalphalipoprotememic HDL (Table 1C). 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).

Clinically, evidence of atheroma was absent, and carotid intima-media thickness in both III.3 and his sister (III.5) was normal. Similarly, there was no record of ischemic CVD in patients 1.2 or II.1, who died from systemic amyloidosis aged 74 and 82 years, respectively.

Aggregation Studies

Predictive analysis of the apoC-III polypeptide in its lipid free state revealed that the Asp25Val mutation enhances both its beta-sheet content and aggregation propensity (Fig. 3A and 3B). Although wild-type and Asp25Val apoC-III initially, showed similar circular dichroic spectra, the mutation appears to cause a transition towards prominent beta-sheet content (Fig. 3C) associated with rapid self-aggregation into typical amyloid fibrils (Fig. 3D).

We compared the 3D NMR structure of the variant Asp25Val-apoC-III with that of the wild type counterpart already characterized in the presence of SDS (9). Two-dimensional 1H,15N NMR spectra showed small (<0.2 ppm, excluding residue 25) amide chemical shift changes in the variant (Fig. 4A and 4B) which were clustered within approximately one helical turn (3.6 residues) of the mutation site. No chemical shift perturbations were observed elsewhere in the sequence. The hydrodynamic radii of wild type and Asp25Val apoC-III micellar complexes, recorded by TRACT relaxation interference experiments, were virtually indistinguishable (Fig. 4C). Moreover, there was no difference in the helical conformation of residues 20-30.

DISCUSSION

Two very recent large population studies confirmed the clinical protection against ischemic CVD resulting from APOC3 deficiency (7,8). Here we report a naturally occurring APOC3 mutation, not previously identified in whole-exome sequencing studies (6-8) causing low levels of plasma TG and apoC-III and a favourable lipoprotein profile in a French kindred affected with severe renal amyloidosis. Concordance between the APOC3 mutation and presence of amyloid among members of this kindred, coupled with identification of Asp25Val apoC-III within amyloid fibrils and in vitro demonstration of its amyloidogenicity, establishes that the Asp25Val variant causes the amyloidosis. 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.

Our in vitro studies clearly show that, despite the remarkable differences in folding dynamics and kinetics of aggregation between wild-type and Asp25Val apoC-III in their lipid free state, their conformation and colloidal stability are similar in the lipid bound state. Wild- type apoC-III was recently reported to self-aggregate into polymeric ordered structures with a peculiar triangular geometry and a Mobius strip conformation after 3 days of shaking in physiological buffer. Although these structures do not resemble the genuine amyloid fibrils that we obtained under physiological conditions with the Asp25Val variant, it is worth noting that aggregation of lipid free wild-type apoC-III was preceded by a gradual structural transition that resembles the conformation rapidly adopted by the Asp25Val variant (Fig. 3C).There is no evidence suggesting in vivo amyloidogenicity of wild-type apoC-III and demonstration of aggregation in vitro does not necessarily imply amyloidogenicity in vivo. Importantly, the absence of wild-type apoC-III within the ex vivo amyloid fibrils from three patients in this kindred, suggests that the wild-type protein does not contribute to amyloid deposition even in the presence of abundant Asp25Val fibrillar seeds, analogous to what we have recently observed in patients with familial p2-microglobulin amyloidosis (14). The preferential incorporation of Asp25Val apoC-III into amyloid fibrils and/or the intracellular degradation of misfolded apoC-III might explain the imbalance in the ratio of wild-type/ Asp25Val apoC-III observed within HDL particles, analogous to amyloidogenic variants of apolipoproteinA-I (26).

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. Since inhibition of LPL-induced lipolysis represents a key physiologic function of apoC-III, rapid VLDL catabolism by LPL may be a consequence of the global reduction of apoC-III concentration in our Asp25Val heterozygotes. The favourable lipid/lipoprotein profile of Asp25Val-carriers is remarkable in the context of CKD and/or ESRD, pathological conditions predisposing to premature atherosclerosis and major ischemic CVD events and commonly associated with an unfavourable lipid profile, an elevated plasma apoC-III concentration and dysfunctional HDL carrying increased amounts of apoCIII, serum amyloid Al and lipoprotein-associated phospho lipase A2 (29-31). Our functional analyses indicate that the cardiovasculo-protective function of HDL is maintained in the uremic Asp25Val proband. Together, the expanded plasma pool of functional HDL particles and the compositional modifications of HDL, such as enrichment in sphingomyelin that can accelerate cellular cholesterol efflux, can endow HDL particles with enhanced intrinsic atheroprotection. Thus, the unexpected favourable lipoprotein profile of our severely uremic Asp25Val-carriers extends the evidence for an important antiatherogenic and cardioprotective role of APOC3 deficiency in ESRD patients.

Since reducing the amyloid fibril precursor protein concentration is known to slow amyloid formation and improve prognosis among patients with a variety of systemic amyloidoses, and since PPARa agonists (fibrates) significantly inhibit hepatic APOC3 transcription (32,33), we postulate that 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).

Table 1

A. Plasma TG, HDL C, and APOC III levels

II.3 III.l 11.3 IU.4 11.5 IV.l IV.2 io IV.4

Asp25Val Asp25Val Asp25¥al Asp25Val Asp25Vil Asp25Val Asp25Val Asp25Val Asp25Val

+ - -/- +/- -/- + - +/- -/- +/- + -

TG 0.30 1.80 0.31 1.1 0.44 034 086 Θ.35 0.50 m

HDL-C ND 0,35 0.71 0,57 0.79 0.59 ND 0.69 0.83 m

ApoC-III ND 54.0 17.7 ND ND ID 44,2 18.8 34.4

(mg/L)

II.3, III. l, III.3, III.4, III.5, IV.l, IV.2, IV.3, IV.4 - refers to individuals in Fig. 1A. Asp25Val +/-, heterozygous for the amyloidogenic apoC III mutation; Asp25Val -/-, no apoC III mutation present. ND, not determined. TG, triglyceride; ApoC III, apolipoprotein C III.

1. Lipoprotein constituents after ultracentrifugation

Protein TG TC PL

mg/tll (%) mg/dl (%) ing/ ll (%) mg/ell (%)

Control

VLDL 12.55 (93) 87.80 (65.0) 8.55 (6.3) 26.26 (19.4) 135.16 roL 4.56 (20.6) 8.45 (38.1) 2.99 (13.5) 6.16 (27.8) 22.16

LDL 82.90 (35.2) 13.72 (7.2) 39.13 (20.4) 71.47 (37.3) 207.22

HDL; 22.79 (47.4) 2.86 (5.9) 8.19 (17.0) 14.26 (29.6) 48.09

HDL.i 93.15 (63.1) 4.63 (3.1) 13.23 (9.0) 36.70 (24.9) 147.71

Proband pil.3)

¥LDL 3.55 (11.4) 20.39 (65.7) 1.60 (5.1) 5.51 (17J) 31.04

IDL 0.42 (4-9) 3.87 (45.9) 1.32 (15.7) 2.82 (33.4) 8.43

LDL 45.80 (31.7) 11.36 (7.9) 30.98 (21.4) 56.53 (39.1) 144.67

HDL: 117.22 (47.5) 5.02 (2.0) 40.19 (16.3) 84.63 (34.3) 247.06

HDL} 62.97 (56.6) 2.79 (2.5) 12.72 (11.4) 32.79 (29.5) 111.27 C, Chemical composition (wt%) of HDL subpoptilations

Subjects HDL2b HDL2a HDL3a HDL3b HDL 3 c

CE (wt%) Proband 29 8 21.1 22.2 27.0 20.1

Control 26,0 23.3 21.6 13.1 12.6

FC (wt%) Proband 7,5 3.1 3.8 6.2 4,2

Control 6.5 4.1 3.3 4.5 1.7

PL (wt%) Proband 31.9 24.4 26.7 31.8 26.6

Control 32.0 25.8 25.6 24.9 16.9

TG ( t%) Proband 2.2 1.4 1.4 2.1 1.9

Control 3.1 2.5 1.9 2.0 1.0

Total protein (wt%) Proband 28.6 49.9 46.0 32.9 47.1

Control 32.4 44.4 47.6 55.4 67.9

Data are shown as wt% of total HDL mass. The proband corresponds to individual III 3

D. Phospholipid and spbingolipid composition (wt%) of total HDL from the proband (III.3) and from a noi molipidemic control subject

PC LPC SM Cer PE PI PS PG PA

Proband 716 0.59 18.1 §.211 1.2 1.1 0.030 0.008 0.013

Control 83.0 0.44 12.9 0 160 11 1.7 0.055 0.006 0.006

Data are shorn as wt% of total phospliolipids + splmigolipids. PC.. phosphatidylcholine; LPC, ^phosphatidylcholine; SM, splmgomyelm; Cer, eeraiiiide; PE, phospktMylethaioiaiime; PI, phospiatidyliaositol; PS, phosphatidylseniie; PG, ptospktidylglyeerol; PA, phosphatide acid. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of identifying a subject having or at risk of having or developing an 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.

2. The method according to claim 1 , wherein the SNV is selected from the group consisting of ApoC3 : c. l34A>T and wherein :

- the presence of the allele (T) of ApoC3::c. l34A>T indicates an increased risk of having or developing hereditary renal amyloidosis.

3. The method according to claim 1 or 2, wherein the sample is a blood.

4. The method according to claim 1 to 3, wherein the presence or absence of said SNV is determined by nucleic acid sequencing or by PCR analysis.

5. A kit suitable for identifying whether a subj ect has or is at risk of having or developing systemic hereditary renal amyloidosis, comprising :

- at least a means for detecting the SNV selected from the group consisting of ApoC3 : c.l34A>T and

- instructions for use.

6. A kit according to claim 5, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a SNV consisting of ApoC3 : c.l34A>T, and

- instructions for use.

7. An APOC3 antagonist for use in the treatment of hereditary renal amyloidosis.

8. The APOC3 antagonist for use according to claim 7, wherein said antagonist is an inhibitor of the APOC3 expression selected from the group consisting of an oligonucleotide sequence targeting the APOC3 gene as an antisense oligonucleotide, siRNA, shRNA, and a ribozyme.

9. The APOC3 antagonist for use according to claim 7, wherein said antagonist is an inhibitor of the APOC3 activity selected form the group consisting of anti-APOC3 antibodies, anti-APOC3 aptamers, and PPAR alpha agonist compound.

10. 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 according to claim 1 to 4.

11. A nuclease for use according to claim 10, wherein the SNV is selected from the group consisting of ApoC3 : c. l34A>T and wherein :

- the presence of the allele (T) of ApoC3::c. l34A>T indicates an increased risk of having or developing hereditary renal amyloidosis.

12. A method for treating a hereditary renal amyloidosis comprising administering to a subject in need thereof a therapeutically effective amount of a APOC3 antagonist, wherein said antagonist is an inhibitor of the APOC3 activity selected form the group consisting of anti-APOC3 antibodies, anti-APOC3 aptamers, and PPAR alpha agonist compound.