Compositions and methods for treating chronic kidney disease associated with a mutation in a terminal complement gene

Abstract

Provided herein are compositions and methods for treating and / or preventing chronic kidney disease (CKD) in a subject harboring a mutation in a terminal complement gene. The disclosed compositions and methods employ an agent capable of modulating, inhibiting, or otherwise affecting the formation and / or activity of the membrane attack complex (MAC), thereby reducing or preventing complement-mediated kidney injury associated with terminal complement gene defects.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CHRONIC KIDNEY DISEASE ASSOCIATED WITH A MUTATION IN A TERMINAL COMPLEMENT GENE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods and compositions for treating and / or preventing chronic kidney disease in subjects having a mutation in a terminal complement gene, using agents capable of affecting formation and / or activity of membrane attack complex (MAC).

[0004] BACKGROUND OF THE INVENTION

[0005] Chronic kidney disease (CKD) is a major cause of morbidity and mortality worldwide with a reported prevalence of up to 13% among adults. CKD can initially be clinically silent and eventually progress to kidney failure, requiring kidney replacement therapy. The underlying causes of CKD often remain unknown owing to a lack of distinguishing clinical characteristics or late clinical presentation.

[0006] The complement cascade, which is an important arm of the innate immune system, has been recognized to play a major role in kidney diseases of different etiologies either as a downstream mediator of inflammation or as a primary driver of kidney injury. The complement system includes numerous soluble proteins that are activated via three distinct pathways: the classical, lectin, and alternative pathways. These pathways converge to form C3 convertases, which catalyze the cleavage of C3 into its active components. This is followed by the activation of subsequent complement proteins, including C5. The larger fragment of C5 (C5b) initiates the terminal complement pathway via the assembly of the membrane attack complex (MAC), a multimeric structure (C5b-9) that integrates into the target cell’s plasma membrane, leading to cellular activation or lysis.

[0007] Dysregulation or over activation of early complement components are known mechanisms of prototypical complement-mediated kidney diseases such as C3 glomerulopathy (C3G) and atypical hemolytic- uremic syndrome (aHUS). These diseases are associated with congenital and acquired defects in the regulation of the alternative pathway. While acquired defects of regulation are mediated by autoantibodies to inhibiting factors such as factor H, or convertase stabilizing antibodies, congenital defects are caused by genetic variants of alternative pathway regulators and activating proteins.

[0008] Terminal complement deficiency is a rare autosomal recessive genetic condition primarily associated with increased susceptibility to Neisseria infections. Studies of patients with terminal complement mutations have largely concentrated on immunological aspects.

[0009] Nevertheless, there is a need in the art for compositions and methods for treating and / or preventing chronic kidney disease in subjects harboring a mutation in a terminal complement gene.

[0010] SUMMARY OF THE INVENTION

[0011] According to some embodiments, there are provided herein compositions and methods for preventing and / or treating chronic kidney disease (CKD) in subjects having a mutation in a terminal complement gene. In particular, the compositions and methods disclosed herein make use of an agent that is capable of modulating / affecting formation and / or activity of membrane attack complex (MAC), in the subjects, thereby preventing or at least partially ameliorating CKD associated with the terminal gene mutation.

[0012] According to some embodiments, without wishing to be bound by any theory or mechanism, the invention is based at least in part on the unexpected and surprising identification of new routes for the initiation and / or progression of kidney diseases, which involves the formation of aberrant complement protein complexes. Such aberrant complexes may arise from genetic mutations in one or more terminal complement system genes. Thus, advantageously the compositions and methods disclosed herein provide a patient-specific, targeted, gene-based therapy to an otherwise unmet need in the prevention of treatment of CKD (i.e., CKD initiation or progression). Otherwise, in the absence of such a targeted approach, afflicted subjects may be treated with general kidney- protective approaches ultimately requiring kidney replacement therapy. Accordingly, advantageously, the disclosed targeted therapies specifically address the underlying cause of disease rather, rather the subsequent non-specific clinical manifestations.

[0013] According to some embodiments, as exemplified herein, a high prevalence which are related to terminal complement deficiency as a cause of kidney failure. In some embodiments, such deficiency may act as a primary cause of kidney disease or as an exacerbating factor in various kidney pathologies, mediated by the formation of aberrant membrane attack complexes.

[0014] According to some embodiments, terminal complement deficiencies are identified at a relatively high prevalence among subjects with kidney failure. In some embodiments, such deficiencies may constitute a primary etiologic factor or may act as an exacerbating co-factor in various kidney pathologies, mediated by the formation of aberrant membrane attack complexes.

[0015] According to some embodiments, there is provided a method of treating and / or preventing a chronic kidney disease (CKD) in a subject in need thereof, the method includes administering to the subj ect an agent capable of at least partially inhibiting activity and / or formation of a membrane attack complex (MAC), wherein the subj ect harbors a mutation in a gene encoding for one or more terminal complement genes.

[0016] According to some embodiments, the terminal complement genes may include: C5, C6, C7, C8 and / or C9 terminal complement gene.

[0017] According to some embodiments, the terminal complement gene includes a C8 terminal complement gene and / or a C9 terminal complement gene.

[0018] According to some embodiments, a mutated C8 gene encodes for a mutated C8A terminal complement protein and / or a mutated C8B terminal complement protein. According to some embodiments, a mutated C8 gene encodes a mutated C8A and / or C8B terminal complement protein.

[0019] According to some embodiments, the subject harbors a mutation in a single terminal complement gene.

[0020] According to some embodiments, the agent is capable of affecting expression and / or function of one or more of C5-C9 terminal complement proteins, involved in the formation or activity of the MAC.

[0021] According to some embodiments, the agent may be a protein, a peptide, an antibody, an antigen binding fragment of an antibody, a small molecule, and / or a nucleic acid.

[0022] According to some embodiments, the agent is an antibody, including a monoclonal antibody selected from: Eculizumab and Ravulizumab.

[0023] According to some embodiments, the agent is an inhibitory peptide, including Zilucoplan.

[0024] According to some embodiments, agent is formulated in a suitable pharmaceutical composition.

[0025] According to some embodiments, there is provided a pharmaceutical composition including an agent capable of at least partially inhibiting activity and / or formation of a membrane attack complex (MAC), for treating and / or preventing a kidney disease in a subject harboring a mutation in a gene encoding for one or more terminal complement genes.

[0026] According to some embodiments, the pharmaceutical composition further includes a suitable excipient.

[0027] Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] Fig- 1- shows a schematic illustration of normal complement cascade and a pathomechanism leading to kidney disease associated with mutated terminal complement genes, according to some embodiments;

[0030] Figs. 2A-C show C8 terminal complement variants (C8A and C8B) in subjects having kidney failure. Fig. 2A: Exon structure of human C8A and C8B cDNA, and domain structure of Complement Factor 8 Alpha and Beta chains (C8A, C8B). The nucleotide change and deduced protein change are highlighted by the red line. ATG start codon and TGA / TAG stop codons are indicated. TSP1- Thrombospondin 1 domain, LDLR- Low Density Lipoprotein Receptor class A domain, MACPF- Membrane Attack Complex / Perforin domain, EGF- Epidermal Growth Factor like domain; Fig. 2B: 3D model of C8A and C8B proteins, with and without the truncating variants. The model was generated by SWISS-MODEL. The N Terminus, upstream to the early stop codon at residue 210 (C8A) or residue 428 (C8B), is indicated in blue. The C Terminus, downstream to the early stop codon is indicated in red; Fig. 2C: Western Blot analysis of patients’ sera showing deficit of terminal complement proteins. The label of each lane indicates the affected gene in each patient, including one healthy unrelated individual as control (labeled WT). C6 was used as loading control.

[0031] Fig. 3 - shows images of histopathology of kidney biopsy obtained from a patient having a C8A truncating variant, exhibiting IgA Nephropathy - Histopathological findings showing glomerular mesangioproliferative disease with in situ complement activation and deposition. The biopsy was taken at the age of 13 years from the patient’s native kidney, light microscopy images (upper panel) and immunofluorescence images (lower panel) are presented. H&E: Hematoxylin and Eosin stain, PAS: Periodic acid Schiff stain, JMS: Jones’ Methenamine Silver stain; and

[0032] Fig. 4 - Tables and graphs showing results of proteomic analysis of MAC components exhibiting aberrant MAC formation. Complexes were analyzed following in vitro complement activation, and pull-down of C6 containing complexes using co-IP. Each sample is compared to the average protein level among controls. While the complete C5-9 MAC is formed in controls, very low levels of C8 and C9 are detected among cases. Abbreviations: co-IP, coimmunoprecipitation; MAC, membrane attack complex.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The principles, uses, and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation. In the figures, same reference numerals refer to same parts throughout.

[0035] According to some embodiments, there are provided herein compositions and methods for treating and / or preventing chronic kidney disease (CKD) in a subject harboring a mutation in a terminal complement gene. The disclosed compositions and methods employ an agent capable of at least partially modulating, inhibiting, or otherwise affecting the formation and / or activity of the membrane attack complex (MAC), thereby reducing or preventing complement-mediated kidney injury associated with terminal complement gene defects.

[0036] As used herein, the term “Terminal complement genes” refers to genes that encode proteins involved in the terminal pathway of the complement system, which is part of the innate immune response. The terminal complement pathway leads to the formation of the membrane attack complex (MAC), a structure that can puncture cell membranes, leading (under normal functioning) to the destruction (lysis) of pathogens, such as bacteria, and infected or dysfunctional cells.

[0037] As used herein, the terminal complement genes may include any one of: C5, C6, C7, C8, C9, any subunits thereof, or any combinations thereof. Each possibility is a separate embodiment.

[0038] Below is a short description of the members of the terminal complement genes and their functioning: The C5 terminal complement gene encodes the C5 protein, which is cleaved into C5a and C5b. C5b initiates the assembly of the MAC by binding to other complement proteins.

[0039] The C6 terminal complement gene encodes the C6 protein, which binds to C5b to form a C5b6 complex.

[0040] The C7 terminal complement gene encodes the C7 protein, which binds to the C5b6 complex.

[0041] The addition of C7 allows the complex to insert into the lipid bilayer of the target cell membrane. C8 (C8A, C8B, C8G genes): The C8 (C8A, C8B, C8G terminal complement genes encode the C8 proteins, which are made up of three polypeptide chains (C8a, C8P, and C8y), respectively. C8 binds to the C5b-7 complex and begins the process of membrane penetration.

[0042] The C9 terminal complement gene encodes the C9 protein, which polymerizes and binds to the C5b-8 complex to form the full MAC. C9 forms a pore in the target cell’s membrane, leading to cell lysis.

[0043] In some embodiments, Wild-type human terminal complement components C5-C9 correspond to the following UniProt accession numbers: human C5 is UniProtP01031, C6 isUniProt P13671, C7 is UniProt P10643, C8A is UniProt P07357, C8B is UniProt P07358, C8G is UniProt Q9Y5Q0, and C9 is UniProt P02748.

[0044] In some embodiments, Wild-type human terminal complement genes correspond to the following gene identifiers: C5 has Entrez Gene ID 727, HGNC symbol C5, and Ensembl ID ENSG00000106804; C6 has Entrez Gene ID 729, HGNC symbol C6, and Ensembl ID

[0045] ENSG00000039537; C7 has Entrez Gene ID 730, HGNC symbol C7, and Ensembl ID

[0046] ENSG00000112936; C8A has Entrez Gene ID 731, HGNC symbol C8A, and Ensembl ID

[0047] ENSG00000157131; C8B has Entrez Gene ID 732, HGNC symbol C8B, and Ensembl ID

[0048] ENSG00000021852; and C9 has Entrez Gene ID 735, HGNC symbol C9, and Ensembl ID ENSG00000113600.

[0049] In some embodiments, Wild-type human terminal complement genes correspond to the following RefSeq mRNA (NM) accession numbers: C5 is represented by NM_001735.3; C6 by NM_000065.5; C7 by NM_000587.4; C8A by NM_000562.3; C8B by NM_000066.2; and C9 by NM_001737.4. These RefSeq accession numbers identify the canonical mRNA transcripts for each terminal complement component and may be used for reference, sequence alignment, mutation annotation, or diagnostic assay design within the context of the present invention.

[0050] As used herein, the term “chronic kidney disease” and “CKD” are interchangeable, and relate to a disease affecting the function of the kidneys. In some embodiments, the CKD relates to the gradual loss of kidney function over time. In some embodiments, the CKD is associated with a mutation in a terminal complement gene. In some embodiments, the CKD is caused by a mutation in a complement terminal gene. In some embodiments, the CKD is induced by a mutation in a complement terminal gene. In some embodiments, the CKD is monogenic (i.e, caused by one or more mutations in a single gene). In some embodiments, the mutation(s) may follow an inheritance pattern, such as autosomal dominant or autosomal recessive and can lead to progressive kidney damage. In some embodiments, the etiology (clinical or histological diagnosis) of the CKD may be selected from: IgA neuropathy, diabetic neuropathy, CKD related to hypertension, CKD of unknown cause, polycystic kidney disease, focal segmental glomerulosclerosis, hemolytic uremic syndrome, MPGN (membranoproliferative glomerulonephritis) or C3 nephropathy and the like, or any combinations thereof. Each possibility is a separate embodiments. In some embodiments, the clinical diagnosis of the terminal complement associated kidney disease includes IgA neuropathy and / or diabetic neuropathy.

[0051] As used herein, the term “mutation” in a gene refers to one or more mutations at any portion of the gene (in particular, the coding region thereof), which may result in a corresponding mutation in the encoded protein. In some embodiments, the mutation may be at any domain of the encoded protein. In some embodiments, the mutation may include any type of mutation, including, for example, substitution, addition, deletion of one or more nucleic acids (e.g. in the encoding gene) or one or more amino acids (in the expressed protein). In some embodiments, the terms “mutated” and “aberrant” may be used interchangeably.

[0052] As used herein, the term “treating” includes, but is not limited to one or more of the following: abrogating, ameliorating, inhibiting, attenuating, blocking, suppressing, reducing, delaying, halting, alleviating and / or preventing symptoms associated with a chronic kidney disease. Each possibility is a separate embodiment.

[0053] Reference is now made to Fig. 1, which shows a schematic illustration of how pathogenic variants in terminal complement genes may affect or cause aberrant MAC formation, which in turn can lead to kidney disease. Illustrated is a normal complement cascade and a pathomechanism leading to kidney disease. In the example shown in Fig. 1, abnormal (mutated) C8 or C9 proteins result in inability of C9 monomers to connect to the forming MAC and the absence of intact membranes penetrating pores. The resulting partial MAC, lacking C9 and some of the C8 chains, may lead to complement deposition in the glomeruli, either by itself as protein aggregates (right) or as part of immune complexes (left).

[0054] According to some embodiments, the mutated terminal complement gene may be any one of C5, C6, C7, C8 or C9. Each possibility is a separate embodiment. In some embodiments, the mutated terminal complement gene is C6. In some embodiments, the mutated terminal complement gene is C7. In some embodiments, the mutated terminal complement gene is C8. In some embodiments, the mutated terminal complement gene is C9. In some embodiments, exemplary C6 mutations may include, for example, but not limited to: missense variants such as p.W114R, p.P521L, p.R596W, and p.Y285C; nonsense variants such as p.R596X and p.Q449X; frameshift variants such as c.821delG and c.1102dupA; and splice-site mutations such as C.1248+1G>T. Each possibility is a separate embodiment.

[0055] In some embodiments, exemplary C7 mutations may include, for example, but not limited to: missense variants such as p.G379R, p.Y497C, p.T166M, and p.R521W; nonsense variants such as p.E356X and p.W389X; frameshift variants such as c.l 135delC and c.210 21 linsG; and splicesite variants such as c.281+2T>C and c.2045-lG>A. Each possibility is a separate embodiment.

[0056] In some embodiments, the mutated terminal complement gene is C8. In some embodiments, the mutation is in C8A and / or C8B. In some embodiments, the mutation is a truncation mutation, leading to the expression of a corresponding truncated protein.

[0057] According to some embodiments, non-limiting examples of mutations in terminal complement genes may include mutations in C8 or C9, such as missense, nonsense, frameshift, splice-site, or deletion variants. Exemplary C8A mutations may include, for example, but not limited to: p.R444H, p.Y387C, p.Q286X, p.W41X, c,195delG, c.843+lG>A, p.Tyr210* (c.630C>A), and exon 3-5 deletions. Each possibility is a separate embodiment. Exemplary C8B mutations may include, for example, but not limited to: p.R95X, p.K325T, p.R461Q, p.W362X, c. H5delA, p.Arg428* (C.1282OT), and C.1629+1G>A. Each possibility is a separate embodiment. Exemplary C9 mutations may include, for example, but not limited to: p.P167S, p.E291K, p.W397X, p.Q267X, c,140delG, p.Cysl l9Gly (c.355T>G), and c,1032+5G>A. Each possibility is a separate embodiment. Any mutation resulting in reduced expression, reduced activity, defective polymerization, or impaired incorporation of a C8 or C9 into the membrane attack complex (MAC) is encompassed herein.

[0058] According to some embodiments, upon identification of a subject harboring a mutation in a terminal complement gene, in order to prevent and / or treat a kidney disease, an agent capable of affecting the formation of an (aberrant) MAC on kidney cells.

[0059] In some embodiments, the agent may at least partially inhibit or otherwise affect activity or expression of one or more terminal complement genes or proteins.

[0060] In some embodiments, the agent is an antibody, or any antigen binding fragment thereof. In some embodiments, the agent is a terminal-complement specific antibody. In some embodiments, the antibody is a monoclonal antibody, capable of binding to a terminal complement protein and prevent or otherwise affect the undesired formation of the MAC. In some exemplary embodiments, the antibody may be Eculizumab and / or Ravulizumab.

[0061] According to some embodiments, the terminal-complement-specific antibody may be selected from, but is not limited to, an anti-C5 antibody (e.g., eculizumab, ravulizumab, crovalimab, tesidolumab, pozelimab, or research clones such as BB5.1), an anti-C6 antibody (e.g., clones 2B7, 9C4, or 7D11), an anti-C7 antibody (e.g., clones WU 3.2, 742, or mAblO4), an anti- C8 antibody (e.g., C8A-specific or C8B-specific monoclonal antibodies such as clone E5), or an anti-C9 antibody (e.g., clone aEl 1 or 5C6). In some embodiments, the antibody is an inhibitory antibody that prevents terminal complement complex assembly or blocks MAC formation. Each possibility is a separate embodiment.

[0062] In some embodiments, the agent is a protein or a peptide, capable of affecting expression and / or activity of a terminal complement gene and / or formation of MAC. In some exemplary embodiments, the peptide may be Zilucoplan.

[0063] According to some embodiments, non-limiting examples of a protein or peptide capable of modulating expression and / or activity of a terminal complement gene product or inhibiting formation of MAC, may include, for example, but not limited to: CD59, factor H, factor I, vitronectin, clusterin, soluble complement receptor 1 (sCRl), CR2-factor H fusion proteins, recombinant CD59, mini-FH proteins, OMCI, compstatin or compstatin derivatives (e.g., Cp40, AMY-101), Zilucoplan, Nomacopan, C5 -binding peptides, C9-inhibitory peptides, and pathogen- derived complement inhibitors (e.g., SCIN-derived peptides, Efb-based peptides, and Salp20- derived peptides). Each possibility is a separate embodiment.

[0064] In some embodiments, the agent is a nucleic acid molecule (such, for example, an antisense, inhibiting RNA (siRNA), miRNA, and the like), capable of affecting expression of a terminal complement gene. In some embodiments, affecting expression of a terminal complement gene is of an aberrant terminal complement gene.

[0065] In some embodiments, the agent is a small molecule, capable of affecting expression and / or activity of a terminal complement gene and / or formation of MAC. In some embodiments, the small molecule may affect expression or activity of an aberrant terminal complement gene.

[0066] According to some embodiments, there is provided a composition (also referred to herein as pharmaceutical composition) which includes the agent capable of affecting formation of MAC, expression of a terminal complement gene, activity of a terminal complement protein. Each possibility is a separate embodiment. In some embodiments, the composition may include one or more suitable excipients, according to the purpose, type and / or use of the composition.

[0067] In some embodiments, excipient is a pharmaceutical excipient which may include or a pharmaceutical carrier, vehicle, buffer and / or diluent. Non-limiting examples of excipients may include buffers (e.g., phosphate, citrate, acetate), tonicity-adjusting agents (e.g., sodium chloride, mannitol, dextrose), stabilizers (e.g., sucrose, trehalose, glycine, PEG), surfactants (e.g., polysorbate 20 or 80), preservatives (e.g., benzyl alcohol, phenol), chelating agents (e.g., EDTA), viscosity modifiers (e.g., HPMC, CMC, PVP), lipids or emulsifiers (e.g., lecithin, cholesterol, poloxamers), fillers or diluents (e.g., lactose, MCC), and lubricants (e.g., magnesium stearate). Each possibility is a separate embodiment.

[0068] According to some embodiments, there is provided a method for treating or preventing a CKD in a subject having a mutated terminal complement gene (and hence a corresponding mutated protein), the method includes administering to the subject a suitable agent capable of affecting formation of a MAC complex, or a composition including the same.

[0069] According to some embodiments, any suitable route of administration to a subject may be used for the agent or the composition of the present invention, including but not limited to, local and systemic routes. Exemplary suitable routes of administration include, but are not limited to: orally, intra-nasally, parenterally, intravenously, topically, enema or by inhalation. According to another embodiment, systemic administration of the composition is via an injection. For administration via injection, the composition may be formulated in an aqueous solution, for example in a physiologically compatible buffer including, but not limited to Hank’s solution, Ringer’s solution, or physiological salt buffer. Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi-dose containers with, optionally, an added preservative.

[0070] According to another embodiment, administration systemically is through a parenteral route. According to some embodiments, parenteral administration is administration intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, intravitreally, or subcutaneously. Each of the abovementioned administration routes represents a separate embodiment of the present invention. According to another embodiment, parenteral administration is performed by bolus injection. According to another embodiment, parenteral administration is performed by continuous infusion. According to some embodiments, preparations of the composition of the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions, each representing a separate embodiment of the present invention. Non-limiting examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

[0071] According to another embodiment, parenteral administration is transmucosal administration. According to another embodiment, transmucosal administration is transnasal administration. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of skill in the art.

[0072] According to another embodiment, compositions formulated for injection may be in the form of solutions, suspensions, dispersions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Non-limiting examples of suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides.

[0073] According to another embodiment, the composition is administered intravenously, and is thus formulated in a form suitable for intravenous administration. According to another embodiment, the composition is administered intra-arterially, and is thus formulated in a form suitable for intra-arterial administration. According to another embodiment, the composition is administered intramuscularly, and is thus formulated in a form suitable for intramuscular administration.

[0074] According to another embodiment, administration systemically is through an enteral route. According to another embodiment, administration through an enteral route is buccal administration. According to another embodiment, administration through an enteral route is oral administration. According to some embodiments, the composition is formulated for oral administration.

[0075] According to some embodiments, oral administration is in the form of hard or soft gelatin capsules, pills, capsules, tablets, including coated tablets, dragees, elixirs, suspensions, liquids, gels, slurries, syrups or inhalations and controlled release forms thereof.

[0076] According to some embodiments, suitable carriers for oral administration are well known in the art. Compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Non-limiting examples of suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose, and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).

[0077] According to some embodiments, solid dosage forms for oral administration may include capsules, tablets, pill, powders, and granules.

[0078] According to some embodiments, the administration may include any suitable administration regime, depending, inter alia, on the medical condition, patient characteristics, administration route, and the like. In some embodiments, administration may include administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, and the like.

[0079] In some embodiments, the administration may be performed in phases. In some embodiments, the dosage may depend on the body weight, age, disease condition, and the like. In some embodiments, the administration may include, for example, weekly IV administration for 4 weeks followed by a maintenance phase, including bi-weekly IV administration. In some embodiments, when the agent is an antibody, the dosage may range from 150-1500mg per administration dose.

[0080] In some embodiments, the therapeutic agent may be administered at a dose of 0.1-100 mg / kg for a protein or antibody, or 0.01-50 mg / kg for a peptide, by IV, SC, IM, oral, mucosal, or sustained-release delivery. The pharmaceutical composition may be formulated with pharmaceutically acceptable excipients, including buffers, stabilizers, tonicity agents, surfactants, or polymeric carriers, and may be provided as a liquid, lyophilized powder, oral tablet, capsule, liposome, nanoparticle, or controlled-release implant.

[0081] In some embodiments, when the agent is a nucleic acid, it may be administered by intravenous, subcutaneous, intramuscular, intrathecal, intraperitoneal, intranasal, oral, transdermal, or topical routes. The nucleic acid may be delivered using lipid nanoparticles, liposomes, polymeric nanoparticles, micelles, exosomes, PLGA particles, electroporation, or a viral vector such as AAV, adenovirus, or lentivirus. Exemplary dosages include 0.001-10 mg / kg for mRNA and RNA-based therapeutics, 0.1-10 mg / kg for plasmid DNA or gene-editing RNPs, and 1 * 10lo-l x 1015vg for viral vector delivery. According to some embodiments, diagnosing a subject suitable for treatment may include identifying a mutation in a terminal complement gene by a genetic assay such as sequencing, PCR- based detection, MLP A, WES, or WGS. Diagnosis may further include assessing complement activity using CH50 or AH50 assays, SC5b-9 ELISA, immunohistochemistry, immunoblotting, or flow cytometry to measure complement deposition or MAC dysregulation.

[0082] According to some embodiments, the agent or composition may be used in combination with other therapeutic agents. The components of such combinations may be administered sequentially or simultaneously / concomitantly in separate or combined pharmaceutical formulations by any suitable administration route. Non-limiting examples of additional / other agents may include, for example, RAAS inhibitors (e.g., ACE inhibitors, ARBs, mineralocorticoid receptor antagonists), SGLT2 inhibitors, immunosuppressive or anti-inflammatory agents, lipid- lowering agents, anti-fibrotic drugs, complement pathway inhibitors (including C3, factor B, factor D, or MASP-2 inhibitors), antihypertensive agents, anticoagulants, gene-targeted nucleic acid therapeutics, or supportive CKD therapies.

[0083] In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated. As used herein, the term comprising includes the term consisting of.

[0084] As used herein, the term “about” may be used to specify a value of a quantity or parameter (e.g. the length of an element) to within a continuous range of values in the neighborhood of (and including) a given (stated) value. According to some embodiments, “about” may specify the value of a parameter to be between 80 % and 120 % of the given value. According to some embodiments, “about” may specify the value of a parameter to be between 90 % and 110 % of the given value. According to some embodiments, “about” may specify the value of a parameter to be between 95 % and 105 % of the given value.

[0085] As used herein, according to some embodiments, the terms “substantially” and “about” may be interchangeable.

[0086] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub- combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced be interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope. The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0087] EXAMPLES

[0088] Methods

[0089] Study population

[0090] A national multicenter prospective study involving dialysis units was used to establish a Kidney Failure Genetic Cohort. A cohort of adult Israeli patients having a common ethnic decent undergoing kidney replacement therapy, mainly dialysis treated were used in the study.

[0091] Clinical assessment and diagnosis

[0092] A single or multiple primary clinical diagnoses were recorded for each participant based on their medical records and their primary clinical nephrology assessment. In addition, all participants underwent a clinical interview at the time of study recruitment, which included a comprehensive review of their medical files, imaging studies and kidney biopsy information when available. The primary kidney clinical diagnoses were classified as either diabetic nephropathy, hypertensive nephropathy, glomerulopathy, polycystic kidney disease, congenital anomalies of the kidney and urinary tract (CAKUT), tubulo-interstitial disease, nephrolithiasis, and CKD of unknown etiology (unknown) or other diagnosis. Furthermore, information regarding the medical history, family history, timing of kidney failure, status of consanguinity, and extra-renal manifestations were obtained.

[0093] Exome sequencing (ES) and variant interpretation

[0094] Exome sequencing was performed according to best practice guidelines. Variant calling and interpretation was performed on the Franklin Platform (Genoox, Tel-Aviv, Israel) according to the American College of Genetic and Genomic Medicine (ACMG) guidelines.

[0095] DNA was isolated from PBMCs using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. Proband ES was performed on genomic DNA for all affected individuals using an IDT xGeb Exome Hyb Panel V2 and Illumina NoveSeq6000 sequencing technology. For each sample, paired end-reads (2>< 100bp) were obtained, processed, and mapped to the genome. The BWA-MEM algorithm Version 0.7.17-rl 188 was used to align the sequence reads to the human reference genome (hg37). The HaplotypeCaller algorithm of GATK V.4.4.0.0 was applied for variant calling, as recommended in the best practice pipeline.

[0096] Variant interpretation

[0097] Exome sequencing data was filtered using the Franklin Platform and interpreted according to the American College of Genetic and Genomic Medicine (ACMG) guidelines. Variants were classified as pathogenic, likely pathogenic, variants of unknown significance (VUS), likely benign or benign. ACMG standards and guidelines recommend 28 criteria for variant pathogenicity classification. The Genoox Classification Engine ran 17 of them automatically. The artificial intelligence-based variant classification engine automates rules PVS1, PSI, PM1, PM2, PM4, PM5, PP2, PP3, PP5, BAI, BS1, BS2, BP1, BP3, BP4, BP6, and BP7. The remaining rules cannot be automated, as they require clinical information specific to the patient genotype, e.g., familial data, de novo evidence (PS2, PM6), segregation data (PPI, BS4), and / or allelic data (PM3, BP2). Therefore, the remaining criteria were manually classified. All variants reported pathogenic or likely pathogenic were evaluated by Clinvar and by the Genoox Classification Engine, all rare (MAF<1%) homozygous variants and all rare (MAF<1%) heterozygous variants found in a CKD panel as previously published.

[0098] CNV were also called using the Franklin platform, utilizing the Al algorithm “Rainbow”, which generates a model for copy number prediction by using a learning set of over 30 sequenced samples from the cohort. It calculates the predicted coverage of each exon based on more than 50 unique predictors, including various exons or regions, ideally from different chromosomes, whose coverage is statistically correlated with that of the exon.

[0099] Complement studies

[0100] Complement Assays: Complement measurements were performed using standard clinically approved techniques. Total complement function was measured using CH50 ELISA assay (MicroVue CH50 Eq EIA, Quidel). Specific complement components and immunoglobulins were detected by commercially available kits using common techniques such as radial immunodiffusion (RID, The Binding Site) or nephelometry (BNII, Siemens).

[0101] Western Blot: 8-10% Bis-Acrylamide separation gel was prepared using 5ml Tris 1.5M pH 8.8, 200pl SDS 10%, 4ml Bis-Acrylamide 40%, lOOpl APS 20%, lOpl TEMED, and DDW to a total of 20ml. 4% Bis-Acrylamide stacking gel was prepared using 2.5ml Tris 0.5M pH 6.8, lOOpl SDS 10%, 1ml Bis-Acrylamide 40%, 50pl APS 20%, lOpl TEMED and DDW to a total of 10ml. Samples were reduced and denatured by Laemmli SDS-Sample Buffer (Bioprep) for 5 min in 95°C. The samples were then separated by electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with 8% skim milk in TBST for 30 min in room temperature. Primary antibody (C6-Invitrogen, PA572500; C7- Invitrogen, PA5-103761; C8A- Abcam, ab273626; C8B- Novus Bio- NBP 185990; C9- Santa Cruz, SC69761) was let to hybridize in 5% BSA solution in TBST (overnight, 4°C). Secondary antibodies (Jackson Immunoresearch 115035062 and 111035144) were incubated in 5% skim milk TBST solution, and color was developed using ECL (Cyanagen). Images were captured using Bio-Rad ChemiDoc MP imaging system, and analyzed using ImageLab software (Bio-Rad).

[0102] Proteomic analysis of membrane attack complexes

[0103] Co-Immunoprecipitation (Co-IP): To isolate membrane attack complexes, the sera was in-vitro activated, followed by C6 targeted Co-IP. 200pl serum was activated using 2pl heat aggregated gamma globulin (Quidel, Al 14) and incubated at 37°C for 90 min. 50pl of Dynabeads Protein G (Invitrogen, 10003D) were washed using 200pl 0.02% PBS Tween. Then, 4.5pg of anti-C6 antibody (Invitrogen, PA572500) was conjugated to the beads (20 min incubation, room temperature). The activated serum was then added to the antibody conjugated beads, and incubated for 120 min, room temperature. The beads were washed with fresh PBS and were taken fresh for further processing for mass spectrometry.

[0104] On-Bead Digestion: The beads were resuspended in 100 pl of Elution Buffer I (2M UREA / 50 mM Tris-HCl pH 7.5 / 1 mM DTT), and proteins were digested by the addition of 0.5 pg of sequencing-grade trypsin (Promega). After 1 hour of incubation at room temperature with shaking, the beads were separated on a magnetic rack, and the supernatant was collected. The beads were then resuspended in 100 pl of Elution Buffer II (2M UREA / 50 mM Tris-HCl pH 7.5 / 5 mM IAA), incubated for 5 minutes, separated again on a magnetic rack, and the resulting supernatant was combined with the first elution. A total of 200 pl of eluate was pooled and incubated overnight at room temperature. The digests were then acidified with 1 pl of trifluoroacetic acid (TFA), purified on C18 StageTips (Pierce), and vacuum dried. The dried peptides were resuspended in 2% acetonitrile / 0.1% TFA prior to LC-MS / MS analysis.

[0105] Liquid chromatography - mass spectrometry (LC-MS) Analysis: Peptides were analyzed by mass spectrometry at the Wohl Institute for Translational Medicine Proteomics Unit, Sheba Medical Center, Israel. Purified peptides were separated using the EASYnLC1200 nano-HPLC system with EasySpray columns (PepMap, 50 cm length, 0.75 pm inner diameter) packed with 2 pm C18 material with a 100 A pore size. Separation was performed using a water-acetonitrile gradient, and peptides were injected into the Q-Exactive HF mass spectrometer (Thermo Scientific) via the EasySpray ionization source. The peptides were loaded with Buffer A (0.1% formic acid) and eluted with a gradient of 5-30% Buffer B (80% acetonitrile / 0.1% formic acid) at a flow rate of 300 nl / min over 100 minutes.

[0106] Mass Spectrometric Acquisition: MS acquisition was performed in data-independent acquisition (DIA) mode under positive-ion conditions. The DIA method included a survey scan at 60,000 resolution from 400 to 1,000 m / z, with an automatic gain control (AGC) target of 3E6 and an injection time of 100 ms. Twenty -five DIA windows, spanning 400-975 m / z, were acquired at 30,000 resolution with an AGC target of 1E6 and a 50 ms injection time. Normalized collision energy was set to 27.

[0107] Mass Spectrometric Raw Data Analysis: The raw MS files were processed using DIA-NN 1.8.1 with global cross-run normalization enabled. The output was filtered at a 1% false discovery rate (FDR) at both the precursor and protein group levels. Protein quantification was performed using the MaxLFQ algorithm as implemented in the diann' R package. Database searching was conducted against the Human UniProt database in library -free mode.

[0108] Statistical analysis

[0109] To assess differences in kidney failure age, consanguinity rate, and the percentage of cases with a family history between genetically solved cases and those without a genetic etiology, an unpaired, two-tailed t-test was used. To explore the potential association between terminal complement pathogenic variants and kidney disease, a minor allele frequency (MAF) in the cohort was compared with that of 399 ethnically matched controls without kidney disease. The genotype frequencies in the cohort did not deviate from Hardy-Weinberg equilibrium (HWE), based on allele frequencies in the control cohort and accounting for an inbreeding coefficient of 0.0238. This was assessed using the Freeman-Halton extension of Fisher's exact test (two-tailed probability)10 13. Subsequently, the association between the risk genotype and kidney failure was assessed by comparing the observed and expected genotype frequencies in cases and controls using the Freeman-Halton extension of Fisher's exact test. Minor allele frequencies between cases and controls were compared using two-sided Z test for two proportions.

[0110] Example 1: Variants (mutations) in C8A and C8B in individuals with kidney failure Four individuals with kidney failure and complement system mutations were identified. The subjects were found to harbor nonsense variants in two terminal complement genes, a patient with C8B variant and patients with C8A variant. Of note, these variants were absent in the homozygous state from healthy individual datasets (such as gnomAD) as well as in ethnically matched controls. The C8A p.Tyr210* is a truncating variant, located upstream to the membrane attack complex / perforin domain (MACPF). This domain is essential for the insertion into the target cell membrane and contributes to the formation of the transmembrane channel that disrupts the cell membrane, leading to cell lysis.

[0111] Fig 2 A shows the exon structure of human C8A and C8B cDNA, and domain structure of Complement Factor 8 Alpha and Beta chains (C8A, C8B). The nucleotide change and deduced protein change are highlighted by the red line. ATG start codon and TGA / TAG stop codons are indicated. TSP1- Thrombospondin 1 domain, LDLR- Low Density Lipoprotein Receptor class A domain, MACPF- Membrane Attack Complex / Perforin domain, EGF- Epidermal Growth Factor like domain.

[0112] Fig. 2B shows a 3D model of C8A and C8B proteins, with and without the truncating variants. The model was generated by SWISS-MODEL. The N Terminus, upstream to the early stop codon at residue 210 (C8A) or residue 428 (C8B), is indicated in blue. The C Terminus, downstream to the early stop codon is indicated in red.

[0113] Fig. 2C shows Western Blot analysis of sera from patients showing deficit of terminal complement proteins. The label of each lane indicates the affected gene in each patient, including one healthy unrelated individual as control (“WT”). C6 terminal complement protein was used as loading control.

[0114] It is noted that the kidney disease progressed rapidly in the identified subjects. Despite medical therapy all reached kidney failure requiring dialysis. The subject later underwent kidney transplantation, however lost the graft due to acute rejection attributed to thrombotic microangiopathy. Subsequently the patient underwent a second kidney transplant which showed chronic rejection and again IgA deposits as well as complement C3 depositions on kidney transplant biopsy. Another patient presented at age nine with elevated creatinine levels and kidney histological findings suggestive of IgA nephropathy due mesangial IgA and C3 deposits. Subsequently, he progressed to kidney failure at the age of 13 years and underwent kidney transplantation. Unfortunately, he required two additional subsequent kidney transplants over a period of 20 years due to acute and chronic transplants rejections. The patient harboring the pathogenic C8B variant (21-753) presented at age 22 with type 2 diabetes mellitus, progressive proteinuria, and no hematuria. Clinically diagnosed with diabetic nephropathy, he did not undergo kidney biopsy and reached kidney failure at the age of 32 years. None of the three patients had a past medical history of infections, either in general or specifically preceding the onset of their kidney disease.

[0115] However, the significantly higher prevalence of terminal complement pathogenic variants identified, and specifically C8A homozygotes in the cohort compared to the expected number based on the minor allele frequency of ethnically matched controls. The genotype frequencies in the cohort did not deviate from Hardy -Weinberg equilibrium (P = 0.557), suggesting no selection bias, but the homozygous genotype was significantly enriched among cases compared to controls (P = 0.040). The minor allele frequency was significantly higher, 3.7% and 2 homozygotes among cases, compared to only 1.38% among controls with no homozygotes (P = 0.041). This enrichment of terminal complement variants in kidney failure patients indicates the involvement in kidney failure. Another patient with kidney failure with a pathogenic homozygous missense variant in the terminal complement gene encoding to the protein C9 (c.355T>G p.Cysl l9Gly, NM_001737.5) was identified.

[0116] Example 2: Histopathology of kidney biopsy obtained from C8A truncating variant, showing IgA Nephropathy

[0117] The biopsy was taken at the age of 13 years from a CKD patient native kidney, having a mutation in C8A terminal complement protein.

[0118] The results are presented in Fig. 3, where both light microscopy and immunofluorescence images clearly exhibit histological features consistent with mesangial IgA deposition.

[0119] Example 3: Terminal complement variants result in aberrant membrane attack complex formation that may mediate immune kidney injury

[0120] To study the causal association between terminal complement variants and kidney injury, the deleterious effect of the variants were confirmed. The presence of each protein component of the complement cascade in the patients’ serum was measured using nephelometry and radial immunodiffusion methods, as well as total hemolytic activity using CH50 assay (Table 1). This showed reduced levels of C8 and C9 proteins in the two participants harboring C8B or C9 pathogenic variants, respectively, and complete absence of C8 protein in the two participants harboring the C8A pathogenic variant. Accordingly, all four cases had low or very low (CH50 levels, indicating impaired ability to activate the complement cascade and complete the MAC formation. Notably, other components of the complement system were unremarkable.

[0121] Table 1: Total serum complement activity (CH50), complement components and immunoglobulins level in patients with kidney failure and pathogenic variants in terminal complement genes

[0122] Western blotting was used to verify the absence of both chains, C8A and C8B, as well as validate the C9 result (shown in Fig. 2C). Indeed, the corresponding protein was absent in all four cases.

[0123] The disease pathomechanism involves the formation of aberrant and incomplete membrane attack complexes due to the inability to connect C9 monomers to the initial C5b-7 complex. In order to detect these partial complexes, co-immunoprecipitation assays were followed by mass spectrometry for affected patients and healthy controls. Specifically, the patients’ sera was activated in vitro and C6-bound complexes were isolated. The results are presented in Fig. 4. Mass spectrometry revealed that in patients’ sera, incomplete membrane attack complexes were formed and were composed only of C5b-7 components (or C5b-8 in the case of C9 variant). In contrast, in controls, the expected complete C5b-9 membrane attack complex was detected.

[0124] This indicates that following complement activation, aberrant membrane attack complexes form in affected patients, which is also demonstrated as the deposition seen on kidney biopsies disclosed above (Fig. 3).

[0125] Collectively, the results demonstrate that aberrant membrane attack complex formation due to aberrant terminal complement genes, mediate kidney damage and can lead to chronic kidney disease.

[0126] Example 4: Treatment of CKD patients having a terminal complement mutation

[0127] CKD patients having a mutation in a terminal complement gene (including C8A, C8B and / or C9) are administered with Eculizumab agent. The administration is performed according to a treatment regime. The regime includes weekly IV administration for 4 weeks (dosage depending on body weight, ranging from 600-900mg Eculizumab), followed by a maintenance phase, including bi-weekly IV administration (dosage depending on body weight and disease condition, ranging from 300-1200mg Eculizumab).

Claims

CLAIMSWhat we claim is:

1. A method of treating and / or preventing a chronic kidney disease (CKD) in a subj ect in need thereof, the method comprising administering to the subject an agent capable of at least partially inhibiting activity and / or formation of a membrane attack complex (MAC), wherein the subject harbors one or more mutations in a gene encoding for one or more terminal complement genes.

2. The method according to claim 1, wherein the terminal complement genes comprise: C6, C7, C8 and / or C9 terminal complement gene.

3. The method according to claim 1 or 2, wherein the terminal complement gene comprises a C8 terminal complement gene and / or a C9 terminal complement gene.

4. The method according to claim 3, wherein a mutated C8 gene encodes for a mutated C8A terminal complement protein and / or a mutated C8B terminal complement protein.

5. The method according to any one of claims 1-4, wherein the subject harbors a mutation in a single terminal complement gene.

6. The method according to any one of claims 1-5, wherein the agent is capable of affecting expression and / or function of one or more of C5-C9 terminal complement proteins, involved in the formation or activity of the MAC.

7. The method according to any one of claims 1-6, wherein the agent comprises a protein, a peptide, an antibody, an antigen binding fragment of an antibody, a small molecule, and / or a nucleic acid.

8. The method according to any one of claims 1-7, wherein the agent is an antibody, comprising a monoclonal antibody selected from: Eculizumab and Ravulizumab.

9. The method according to any one of claims 1-7, wherein the agent is an inhibitory peptide, comprising Zilucoplan.

10. The method according to any one of claims 1-9, wherein the agent is formulated in a suitable pharmaceutical composition.

11. A pharmaceutical composition comprising an agent capable of at least partially inhibiting activity and / or formation of a membrane attack complex (MAC), for treating and / orpreventing a kidney disease in a subject harboring one or more mutations in a gene encoding for one or more terminal complement genes.

12. The pharmaceutical composition according to claim 11, wherein the terminal complement genes comprise: C6, C7, C8 and / or C9 terminal complement gene.

13. The pharmaceutical composition according to claim 11 or 12, wherein the terminal complement gene comprises a C8 terminal complement gene and / or a C9 terminal complement gene.

14. The pharmaceutical composition according to claim 13, wherein a mutated C8 gene encodes for a mutated C8A terminal complement protein and / or a mutated C8B terminal complement protein.

15. The pharmaceutical composition according to any one of claims 11-14, wherein the subject harbors a mutation in a single terminal complement gene.

16. The pharmaceutical composition according to any one of claims 11-15, wherein the agent is capable of affecting expression and / or function of one or more of C5-C9 terminal complement proteins, involved in the formation or activity of the MAC.

17. The pharmaceutical composition according to any one of claims 11-16, wherein the agent comprises a protein, a peptide, an antibody, an antigen binding fragment of an antibody, a small molecule, and / or a nucleic acid.

18. The pharmaceutical composition according to any one of claims 11-17, wherein the agent is an antibody, comprising a monoclonal antibody selected from: Eculizumab and Ravulizumab.

19. The pharmaceutical composition according to any one of claims 11-17, wherein the agent is an inhibitory peptide, comprising Zilucoplan.

20. The pharmaceutical composition according to any one of claims 11-19, wherein the pharmaceutical composition comprises a suitable excipient.

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