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WO2025110984A1 - Compositions et méthodes pour le traitement de la maladie de wilson - Google Patents

Compositions et méthodes pour le traitement de la maladie de wilson Download PDF

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Publication number
WO2025110984A1
WO2025110984A1 PCT/US2023/031730 US2023031730W WO2025110984A1 WO 2025110984 A1 WO2025110984 A1 WO 2025110984A1 US 2023031730 W US2023031730 W US 2023031730W WO 2025110984 A1 WO2025110984 A1 WO 2025110984A1
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atp7b
liver
promoter
copper
expression
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Robert Kruse
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Hydrogene Therapeutics Inc
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Hydrogene Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine

Definitions

  • BACKGROUND Wilson's disease is a genetic disorder associated with mutation of the ATP7B gene, which disrupts copper metabolism such that excess copper builds up in the afflicted individual's body.
  • copper is absorbed in the small intestine via enterocyte uptake by hCTR1, and copper transport into the blood is mediated by ATP7B at the basolateral aspect of duodenal epithelia. Copper is then conveyed by portal circulation to the liver, where excess copper is removed by excretion into the bile at the apical aspect of hepatocytes, a process that is disrupted by mutations in the ATP7B gene.
  • Wilson's disease generally presents with neurological and/or liver-related symptoms, and while symptoms usually begin between the ages of 5 and 35 years, both early onset (e.g., infancy) and late onset (e.g., > 70 years old) presentations have been documented. Diagnosis may be difficult and often involves a combination of blood tests, urine tests and/or liver biopsy. Neurological symptoms may include tremors, muscle stiffness, trouble in speaking, personality changes, anxiety, and psychosis, while liver-related symptoms may include vomiting, weakness, fluid buildup in the abdomen, swelling of the legs, yellowish skin and itchiness. Complications of Wilson's disease may include increased chances of liver failure, liver cancer, and/or kidney damage.
  • Wilsons disease is a monogenic, autosomal recessively inherited condition associated with mutation of a copper-transporting P-type ATPase (i.e., ATP7B). For a person to be affected, they must inherit a mutated copy of the gene from both parents. More than 500 ATP7B mutations have now been identified, including missense mutations, small deletions/insertions in the coding region, or splice junction mutations. Genetic testing may be used to screen family members of those affected. Early estimates suggested that Wilson's disease occurs at a frequency of about 1 in 30,000 people; however, more recently it has been suggested that the frequency is much higher (e.g., 1 in 7,000 people).
  • Wilson's disease is typically treated with dietary changes (e.g., a low copper diet) and medication (e.g., chelating agents such as trientine and d-penicillamine, and also zinc supplements).
  • medication e.g., chelating agents such as trientine and d-penicillamine, and also zinc supplements.
  • chelating agents such as trientine and d-penicillamine, and also zinc supplements.
  • the present disclosure is based, at least in part, on the surprising discovery that non-viral modification of hepatocytes with ATP7B gene in Wilson’s Disease (WD) liver can lead to expansion of those hepatocytes through a proliferative advantage.
  • WD Wilson’s Disease
  • the present disclosure provides a method of treating of Wilson’s Disease, including the steps of: a) administering a non-viral DNA vector capable stably expressing human ATP7B into liver cells by hydrodynamic injection, and b) yielding a selective proliferative advantage of liver cells harboring the DNA vector in a Wilson’s Disease individuals’ liver over time increasing the amount of those liver cells, wherein no other exogenous agents or chemicals or partial hepatectomy are needed to induce this proliferative advantage.
  • the liver cells are hepatocytes and cholangiocytes.
  • the non-viral DNA vector is integrated into host hepatocytes or cholangiocytes with the use of a transposon system for replication with mitosis.
  • the transposon system is a piggyBac transposon or Sleeping Beauty Transposon.
  • non-viral DNA vector alternatively harbors sequence elements to enable episomal replication for replication with mitosis.
  • the episomal replication sequence is a scaffold/matrix attachment region sequence.
  • the administered vector is double-stranded circular or linear DNA.
  • non-viral DNA vector is at least a promoter, a 5’ UTR, a human ATP7B coding sequence, a 3’ UTR, an enhancer and polyadenylation sequence.
  • the promoter is selected from the group consisting of a hepatocyte-specific promoter, consisting of alpha-1 antitrypsin, human thyroxine binding globulin, hemopexin, albumin, and HBV core promoters.
  • the promoter is selected from the group consisting of a cholangiocyte-specific promoter, consisting of cytokeratin-17, cytokeratin-19, cyclooxygenase-2 (COX-2), midkine (MK), mucin-1 (MUC1), and osteopontin.
  • the promoter is a tandem of a hepatocyte- and cholangiocyte- specific promoter, thereby allowing ATP7B expression in both hepatocytes and cholangiocytes.
  • a promoter is selected that has expression in both hepatocytes and cholangiocytes, such as cytokeratin-18 promoter.
  • the enhancer element is added to the promoter, consisting of a liver-specific enhancer such as human apolipoprotein hepatic control region, human albumin enhancer, human ApoE enhancer, or a viral enhancer such as SV40 enhancer, HBV enhancer I, HBV enhancer II to drive more potent expression.
  • non-viral DNA vector optionally contains at least one intron selected from SV40 intron, Minute Virus of Mice (MVM) intron, and human growth hormone (HGH) intron, preferably in the 5’ UTR to enhance ATP7B expression.
  • the coding sequence for ATP7B is codon-optimized for human hepatocyte expression.
  • the 3’ UTR is selected among human beta-globin UTR, human alpha-globin UTR, or albumin UTR.
  • the non-viral DNA vector does not induce overexpression toxicity of ATP7B in large animals compared to rodents when utilizing constitutively active liver- specific promoters
  • hydrodynamic injection of plasmid DNA occurs through the biliary tract, hepatic vein, or hepatic artery to mediate delivery into liver cells.
  • hepatocytes expressing ATP7B after non-viral delivery will expand at least 2-fold, 3-fold, 4-fold, or 5-fold or more after at least 2 months.
  • the disclosure provide a method of treating a human subject having Wilson’s Disease, comprising administering a non-viral DNA vector into liver cells by hydrodynamic injection, wherein a therapeutically effective dose of the non-viral DNA vector is administered to the human subject via the biliary system into a liver of the human subject.
  • the administration step occurs via an endoscopic retrograde cholangio-pancreatography procedure.
  • the non-viral DNA vector achieves expression in at least 20% of hepatocytes in the human subject after delivery, as detectable by protein or RNA staining.
  • the non-viral DNA vector dose is at least about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg of DNA per kilogram of liver weight of the human subject.
  • the subject exhibits normalization of their liver copper content and restoration of ceruloplasmin levels.
  • the human subject is administered with a copper chelation or zinc therapy prior to administration of the non-viral DNA vector.
  • the hepatic pathology of the human subject is normalized and disruption of the efficiency of biliary hydrodynamic gene delivery is avoided.
  • the copper chelation therapy is selected for D-penicillamine (DPA) or trientine (TETA).
  • DPA D-penicillamine
  • TETA trientine
  • the human subject possesses normal ALT and AST liver enzymes.
  • the human subject possesses liver enzyme elevations within 3 times the upper limit of normal, to assume close to normal liver histology.
  • the human subject have elevated liver enzymes and still require treatment, then the human subject requires a DNA dose at least 50 mg per kilogram of liver weight to compensate for distorted pathology.
  • the human subject has fulminant hepatitis induced by Wilson’s Disease and is administered via an endoscopic procedure with an elevated non-viral DNA dose of at least 50 mg per kg of liver weight to compensate for distorted pathology.
  • the efficacy of the non-viral DNA vector is monitored with urinary copper, serum copper, ceruloplasmin, and/or biopsy-gained hepatic copper measurements.
  • the subject is preferentially selected among patients with Wilson’s disease who do not tolerate current copper chelation medications, or alternatively have neurological disease with any treatment response, or alternatively have antibodies against AAV capsids.
  • the patient may start or continue taking copper chelation medications and zinc for a period of at least 1-month, at least 2 months, at least 3 months, or at least 6 months to help accelerate the de-coppering process from the body, before cessation of pharmacologic therapy.
  • the combination therapy will prevent hepatocyte turnover from copper-induced death before the genetic treatment has time to take effect.
  • the non-viral DNA vector is maintained either as an episome without integration, or alternatively is facilitated with integration.
  • the human subject is redosed with episomal vectors configured for redosing at least once every one year, at least every two years, at least every three years, or at least every five years.
  • the need for redosing is determined by assessing the elevation in the ALT and AST liver enzymes, such that enzymes fall outside the upper limit of normal.
  • the non-viral DNA is integrated in the genome of the human subject, optionally via a transposase, a large serine recombinase, or a CRISPR-directed homologous recombination.
  • the non-viral DNA vector yields a selective proliferative advantage of liver cells harboring the DNA vector in a Wilson’s Disease individuals’ liver over time increasing the amount of those liver cells positive for the vector-derived ATP7B, optionally wherein no other exogenous agents or chemicals or partial hepatectomy are needed to induce this proliferative advantage, optionally wherein the proliferative advantage can be slowed through the administration of copper chelation and zinc as desired.
  • hepatocytes and cholangiocytes of the liver are targeted for expression with ATP7B.
  • the non-viral DNA vector is integrated into host hepatocytes or cholangiocytes for replication with mitosis to provide stability due to the proliferative advantage and/or turnover of un-transfected hepatocytes, optionally via a transposon system.
  • the transposon system is a piggyBac transposon, a hyperactive piggyBac transposon, or a Sleeping Beauty Transposon.
  • the non-viral DNA vector alternatively harbors sequence elements to enable episomal replication for replication with mitosis.
  • the episomal replication sequence is a scaffold/matrix attachment region sequence.
  • the administered non-viral vector is a double-stranded circular or linear DNA.
  • the non-viral DNA vector includes a promoter, a 5’ UTR, a human ATP7B coding sequence, a 3’ UTR, an enhancer and polyadenylation sequence.
  • the promoter is selected from the group consisting of a hepatocyte-specific promoter, consisting of alpha-1 antitrypsin, human thyroxine binding globulin, hemopexin, albumin, LP1, P3, and mouse transthyretin promoters.
  • the promoter is selected from the group consisting of a cholangiocyte-specific promoter, consisting of cytokeratin-17, cytokeratin-19, cyclooxygenase-2 (COX-2), midkine (MK), mucin-1 (MUC1), and osteopontin.
  • the promoter is a tandem of a hepatocyte- and cholangiocyte- specific promoter, with the cholangiocyte 5’ in order to the hepatocyte promoter, thereby allowing ATP7B expression in both hepatocytes and cholangiocytes.
  • a promoter is selected that has expression in both hepatocytes and cholangiocytes, such as cytokeratin-18 promoter, or the alpha-1 antitrypsin promoter.
  • the enhancer element is added to the promoter, consisting of a liver-specific enhancer such as human apolipoprotein hepatic control region, human albumin enhancer, human ApoE enhancer, or a viral enhancer such as HBV enhancer I, HBV enhancer II to drive more potent expression.
  • non-viral DNA vector optionally contains at least one intron selected from SV40 intron, Minute Virus of Mice (MVM) intron, and human growth hormone (HGH) intron, preferably in the 5’ UTR to enhance ATP7B expression.
  • the coding sequence for ATP7B is codon-optimized for human hepatocyte expression, such that the expression level is at least 2-fold higher than the wild-type ATP7B sequence.
  • the 3’ UTR is selected among human beta-globin UTR, human alpha-globin UTR, or a doublet of those sequences for added stability.
  • the non-viral DNA vector does not induce overexpression toxicity of ATP7B in large animals compared to rodents when utilizing constitutively active liver- specific promoters due to differences in delivery efficiency, such that no native regulatory elements are required.
  • expression of ATP7B is constitutive and thus can suppress the endogenous mutant ATP7B, which is regulated by copper levels, thereby enhancing the therapeutic effect by alleviating the malfunctioning form inside the cell.
  • the native ATP7B promoter, or other promoter controlled by metal-responsive elements are preferably not used in the viral vector to avoid promoter competition with the endogenous mutant proteins.
  • the subject is further administered with copper supplements if vector expression from ATP7B be too significant and cause copper deficiency
  • the native ATP7B mutant’s expression is knocked down with shRNA that is also encoded on the non-viral vector, such that the new delivered wildtype ATP7B avoids mispairing with the mutant receptor inside the cell.
  • hydrodynamic injection of DNA occurs alternatively through the hepatic vein, or hepatic artery to mediate delivery into liver cells.
  • hepatocytes expressing ATP7B after non-viral delivery will expand at least 2-fold, 3-fold, 4-fold, or 5-fold or more after at least 2 months post-gene delivery compared to their original number post-injection.
  • the ATP7B protein coding sequence can tolerate an N-terminal or C-terminal additions for protein identification without affecting protein function.
  • the subject is a canine subject who possesses a pathologic mutation in ATP7B and elevated hepatic copper levels causing silent or active hepatitis.
  • the canine subject will receive wildtype canine ATP7B (NM_001025267.1) into the canine liver.
  • the canine subject will preferentially receive integrative vector strategies to avoid the need for future redosing.
  • Definitions To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below: Unless specifically stated or obvious from context, as used herein, the term “about” is understood as being within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, polypeptide, or fragments thereof.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a Wilson's disease phenotype).
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • an ATP7B analog is meant a molecule that is not identical but has analogous functional or structural features.
  • an ATP7B analog retains the biological activity of a corresponding naturally occurring ATPase, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance or half-life, without altering, for example, copper transport.
  • An analog may include an unnatural or synthetic amino acid or altered amino acid sequences within the ATP7B protein.
  • combination therapy embraces the administration of a gene therapy protocol and one or more additional therapeutic agents (e.g., copper chelating compounds) as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected).
  • “Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous or overlapping manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject, for example, one or more copper chelating compounds while administering a gene therapy protocol as disclosed herein.
  • Sequential or substantially simultaneous administration of each therapeutic agent can be affected by any appropriate route including, but not limited to, oral routes, intravenous routes, sub-cutaneous routes, intramuscular routes, direct absorption through mucous membrane tissues (e.g., nasal, mouth, vaginal, and rectal), and ocular routes (e.g., intravitreal, intraocular, etc.).
  • the therapeutic agents can be administered by the same route or by different routes.
  • one component of a particular combination may be administered by intravenous injection (e.g., a gene therapy protocol) while the other component(s) (e.g., one or more copper chelating compounds) of the combination may be administered orally.
  • the components may be administered in any therapeutically effective sequence.
  • phrase “combination” embraces groups of compounds and/or non-drug gene therapies useful as part of a combination therapy as disclosed herein.
  • “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • control is meant a standard or reference condition.
  • disease is meant any condition or disorder (e.g., Wilson's disease) that damages or interferes with the normal function of a cell, tissue, or organ.
  • effective amount is meant the amount required to ameliorate the symptoms of a disease (e.g., neurological or liver symptoms of Wilson's disease) relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an effective amount.
  • the effective amount may also refer to levels of gene expression (e.g., ATP7B mRNA or protein expression) in the appropriate tissues of a patient.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more nucleotides or amino acids.
  • a “gene therapy composition” is to be understood as meaning a DNA composition (e.g., including a full length ATP7B nucleotide sequence, or portion thereof) for generating prophylaxis and/or treatment of Wilson's disease.
  • gene therapy compositions are medicaments which comprise a full length ATP7B nucleotide sequence, or portion thereof, and are intended to be used in humans or animals for generating prophylaxis and/or treatment of Wilson's disease.
  • “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • isolated polynucleotide is meant a nucleic acid molecule (e.g., a DNA, an mRNA, a cDNA, and the like) that is free of the genes from which, in the naturally occurring genome of the organism, the nucleic acid molecule of the disclosure is normally associate or derived.
  • the term therefore includes, for example, a recombinant DNA (e.g., including a genomic DNA or cDNA coding for a ATP7B gene, as well as associated regulatory components such as, for example, an enhancer(s), a promoter, 5' and/or 3' untranslated regions (UTRs), and the like) that may be incorporated into: a vector, or an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or into a polynucleotide that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion, or a naked DNA construct such as a plasmid or cosmid or linear DNA) independent of other sequences.
  • a recombinant DNA e.g., including a genomic DNA or cDNA coding for a ATP7B gene, as well as associated regulatory components
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the disclosure.
  • An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. “Mutation” for the purposes of this disclosure means a DNA sequence found in the ATP7B gene of a patient that does not correlate with an established wildtype ATP7B gene sequence, and such mutations may be due to one or more single nucleotide polymorphisms, one or more deletions or insertions of one or more nucleotides, and deletion or insertion of splice site junctions.
  • “Mutation” may also refer to patterns in the sequence of RNA from a patient that are not attributable to expected variations based on known information for the ATP7B gene and are reasonably considered to be novel variations in, for example, the splicing pattern of the ATP7B gene of the patient.
  • the term “or” is understood to be inclusive.
  • the terms “a,” “an,” and “the” are understood to be singular or plural.
  • the term “patient” or “subject” refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.
  • Pharmaceutically acceptable refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • “Pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • a “pharmaceutically acceptable salt” of pooled tumor specific neo-antigens as recited herein may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication.
  • Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH2)n-COOH
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium.
  • pharmaceutically acceptable salts for the pooled tumor specific neo-antigens provided herein, including those listed by Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.1418 (1985).
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease or condition (e.g., Wilson's disease) in a subject, who does not have, but is at risk of or susceptible to developing the disease or condition (e.g., Wilson's disease).
  • “Primer set” means a set of oligonucleotides that may be used, for example, for PCR.
  • a primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers. Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • “nested sub-ranges” that extend from either end point of the range are specifically contemplated.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • reference is meant a standard or control condition.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison (e.g., a wildtype ATP7B gene sequence; Genebank sequence NM_000053).
  • a reference sequence may be a subset of, or the entirety of, a specified sequence; for example, a segment of a full-length cDNA or genomic sequence, or the complete cDNA or genomic sequence.
  • the length of the reference polypeptide sequence will generally be at least about 10-5,000 amino acids, 10-4,000 amino acids, 10-3,000 amino acids, 10-2,000 amino acids,10-1,500 amino acids, 10- 1,000 amino acids, 10-500 amino acids, or 10-100 amino acids.
  • the length of the reference polypeptide sequence may be at least about 10-50 amino acids, more preferably at least about 10-40 amino acids, and even more preferably about 10-30 amino acids, about 10-20 amino acids, about 15-25 amino acids, or about 20 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, about 60 nucleotides, about 75 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1000 nucleotides, about 1250 nucleotides, about 1500 nucleotides, about 1750 nucleotides, about 2000 nucleotides, about 2250 nucleotides, about 2500 nucleotides, about 2750 nucleotides, about 3000 nucleotides, about 3250 nucleotides, about 3500 nucleotides, about 3750 nucleotides, about 4000 nucleotides, about 4250 nucleotides, about 4500 nucleotides, about 4750 nucleotides, about 5000 nucleotides, about 5250 nucleotides, about 5
  • Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide (e.g., an ATP7B polypeptide) of the disclosure, or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity (e.g., 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%). Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., an ATP7B gene described herein), or portions thereof, under various conditions of stringency. (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, more preferably of at least about 42°C, and even more preferably of at least about 68°C.
  • wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid or nucleotide sequence (for example, any one of the amino acid or nucleotide sequences described herein).
  • a reference amino acid or nucleotide sequence for example, any one of the amino acid or nucleotide sequences described herein.
  • such a sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 100% identical at the amino acid sequence or nucleic acid sequence used for comparison (e.g., wildtype ATP7B).
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
  • the terms “treat,” “treated,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., Wilson's disease). It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.
  • the term “therapeutic effect” refers to some extent of relief of one or more of the symptoms (e.g., neurological, liver-related, etc.) of Wilson's disease or its associated pathology.
  • “Therapeutically effective amount” as used herein refers to an amount of an agent or combination therapy which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with Wilson's disease, reducing one or more signs or symptoms of Wilson's disease, preventing or delaying onset of symptoms of Wilson's disease, and the like, beyond what would be expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” agent or combination therapy required.
  • the pharmaceutical compositions typically should provide a dosage of from about 0.0001 mg to about 1500 mg of compound per day, preferably 50 mg to about 1500 mg of compound per day.
  • dosages for systemic administration to a human patient can range from about 0.01-10 ⁇ g/kg, about 20-80 ⁇ g/kg, about 5-50 ⁇ g/kg, about 75-150 ⁇ g/kg, about 100-500 ⁇ g/kg, about 250-750 ⁇ g/kg, about 500-1000 ⁇ g/kg, about 1-10 mg/kg, about 5-50 mg/kg, about 25-75 mg/kg, about 50-100 mg/kg, about 100-250 mg/kg, about 50-100 mg/kg, about 250-500 mg/kg, about 500-750 mg/kg, about 750-1000 mg/kg, about 1000-1500 mg/kg, about 1500-2000 mg/kg, about 5 mg/kg, about 20 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 250 mg/kg, about 300 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450 mg/kg, about 500 mg/kg, about 550
  • Pharmaceutical dosage unit forms are prepared to provide from about 50 mg to about 1500 mg, for example from about 100 to about 1000 mg of the compound or a combination of essential ingredients per dosage unit form.
  • “Full-length Human ATP7B promoter nucleic acid molecule” is meant a polynucleotide encoding an ATP7B promoter. The following sequence starts at -895 before the transcription start site of human ATP7B and extends to +175 of transcription. This sequence is designed to stop after the final Sp1 binding site of the ATP7B promoter, containing a portion of the native ATP7B 5’ UTR.
  • a full-length human ATP7B promoter nucleic acid molecule has at least about 100% identity, 99% identity, 98% identity, 97% identity, 96% identity, 95% identity, 94% identity, 93% identity, 92% identity, 91% identity, or 90% identity to the following nucleic acid molecule sequence: acaaggaaggccatttgcccgcaaaatttagctacactggacgggcaagtacccctacagaaga gaaaacgtctgtgagcccacacgaccggctgctcacctcaacaacttgcacaggcaccagctcc ttcgcggccgccatcttccgccgacccccgaactcaggaaacgcttcactttccttttcct attggctcctgagaaagcaagccgtcccccct attggctctgagaaa
  • a truncated human ATP7B promoter nucleic acid molecule has at least about 100% identity, 99% identity, 98% identity, 97% identity, 96% identity, 95% identity, 94% identity, 93% identity, 92% identity, 91% identity, or 90% identity to the following nucleic acid molecule sequence: cacgaccggctgctcacctcaacaacttgcacaggcaccagctcctttcgccggccgccatctt cccgcgacccccgaactcaggaaacgcttcactttccttttccctattggctcctgagaaagca agccgtgcccccccacgggccaattgtgcgttactattggtttactggtagccgcttc ccacgg
  • an ATP7B promoter enhancer element nucleic acid molecule has at least about 100% identity, 99% identity, 98% identity, 97% identity, 96% identity, 95% identity, 94% identity, 93% identity, 92% identity, 91% identity, or 90% identity to the following nucleic acid molecule sequence: cacgaccggctgctcacctcaacaacttgcacaggcaccagctcctttcgccggccgccatctt ccgcgacccccgaactcaggaaacgcttcactttccttttccctattggctcctgagaaagca agccgtgctcgccccccacgggccaatt
  • SV40 intron sequence nucleic acid molecule is meant a polynucleotide encoding an ATP7B promoter enhancer element.
  • a SV40 intron sequence nucleic acid molecule has at least about 100% identity, 99% identity, 98% identity, 97% identity, 96% identity, 95% identity, 94% identity, 93% identity, 92% identity, 91% identity, or 90% identity to the following nucleic acid molecule sequence: CTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCT CTCTTTTAGATTCCAACCTTTGGAACTGA
  • ATP7B copper-transporting ATPase 2 isoform A polypeptide is meant a polypeptide or fragment thereof having at least about 100% amino acid identity, 99% amino acid identity, 98% amino acid identity, 97% amino acid identity, 96% amino acid identity, 95% amino acid identity, 94% amino acid identity, 93% amino acid identity, 92% amino acid identity, 91% amino acid identity, or 90% amino acid identity to NCBI Reference Sequence: NP_000044.2, representing: MPEQERQITAREGASRKILSKLSLPTRAWEPAM
  • Example of sequence of different polyadenylation sites – SV40 polyadenylation sequence is provided ATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAA CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTT TTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCCGATAAGGATCGATCCGGGC 9.
  • the interferon-beta scaffold/matrix attachment region is provided below.
  • Example of stabilizing 3’UTR example of albumin sequence is provided CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGC TTATTCATCTGTTTTTCTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTT CTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCT 11.
  • pT-LP1-ATP7B Transposon expressing ATP7B under hepatocyte-specific expression TTAACCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTT TCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGT GAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAA ATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTTAT TTCATGTTCTACTTACGTGATAACTTATTATATATTTTCTTGTGGAGGGGCTAGCTCGTG ACCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTG ACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCG ACCCCTTGGAATTTCGGTGGAATTTCGGTG
  • FIGS.1A-1C depict construction and functional validation of plasmid expressing hATP7B with C9-tag.
  • FIG. 1A shows a C9-tag, corresponding to the carboxy-terminal 9 amino acids of bovine rhodopsin was added to the carboxy-terminus of human ATP7B located in the intracellular surface.
  • FIG. 1B shows the gene, ATP7B,C9, was cloned into a plasmid DNA vector, pT-LP1, with a liver-specific LP1 promoter driving expression between the terminal repeats of piggyBac transposon, which facilitate integration. SV40 polyadenylation sequence is not shown.
  • Total pDNA size is 8.6 kB, including the bacterial backbone.
  • FIG.1C shows the intracellular movement of hATP7B,C9 in response to different copper levels (basal, low, and high) inside cells was examined.
  • antibody staining for ATP7B and C9 tag (1D4 antibody) efficiently co-localized, with ATP7B,C9 moving out of the trans-golgi network (TGN) in response to high copper levels.
  • FIG.1D shows ATP7B,C9 copper transporting activity was examined in cells in cells lacking native ATP7B expression.
  • FIGS.2A-2E show the results of hydrodynamic tail vein injection of pT-LP1-hATP7B,C9 results in reduction of liver injury and hepatic copper content in WD mice.
  • FIG. 2A shows the experimental plan, wherein female WD mice were hydrodynamically injected with 1 ⁇ g of pT- LP1-hATP7B,C9 and 1 ⁇ g pCMV-hyPBase, the latter for integration. Heterozygous mice were also injected as a control, and all mice were bleed and harvested at 20 weeks for further analysis. Serum obtained at 20 weeks for un-injected heterozygous mice, un-injected ATP7B KO mice, and treated ATP7B KO mice were compared for ALT (FIG.2B), AST (FIG.2C), and LDH (FIG.2D).
  • FIG.2E shows that the hepatic copper content was measured in the same mice, with significant reduction in the treatment group.
  • FIGS.3A-3D depict hydrodynamic tail vein injection achieves transfection of hepatocytes with ATP7B,C9 without significant expansion over time.
  • FIG. 3A shows immunohistochemical staining for ATP7B,C9 for treated WD mice reveals positive hepatocytes with variable morphology and staining intensity.
  • FIG.3C shows that 10 ⁇ g of reporter plasmid, pCMV-GFP, was injected into WD and Het mice by HTVI, and the % stained area compared at 3 days post-injection.
  • FIG.3D shows H&E staining for female heterozygous, KO untreated, and KO treated mice are depicted, showing minimal differences. Statistical significance was calculated with unpaired, parametric t-tests, * p ⁇ 0.05, ** p ⁇ 0.005, *** p ⁇ 0.0005.
  • FIGS. 4A-4E show that biliary hydrodynamic injection can successfully mediate hATP7B,C9 expression in pig liver.
  • FIG.4A shows that ERCP can be used to access the common hepatic duct, with fluoroscopy verifying catheter position and branching into the liver prior to injection.
  • FIG.4B demonstrates that harvested pig liver shows no abnormalities from injection and no rupture of bile ducts. Sampling of the right lateral, right medial, left medial, and later lateral liver lobes was conducted.
  • FIG. 4C shows the immunohistochemistry for C9-tag in pig liver reveals abundant hATP7B,C9 positive hepatocytes, which were located across all three zones. Rare liver lobules had transfection that exceeded over 80% of hepatocytes.
  • FIG.4A shows that ERCP can be used to access the common hepatic duct, with fluoroscopy verifying catheter position and branching into the liver prior to injection.
  • FIG.4B demonstrates that harvested pig liver shows no abnormalities from injection and no rupture of bile ducts. Sampling of the right lateral, right
  • FIG.4D shows immunofluorescent staining for ATP7B,C9 in pigs demonstrates localization of ATP7B,C9 inside pig hepatocytes. This pattern was similar to ATP7B staining for native pig ATP7B.
  • FIG.4E shows quantification of immunostaining in pigs dosed at 10 mg pDNA. Percent area was calculated among liver lobules, with 8 liver lobules averaged.
  • FIGS.5A-E shows serum chemistries reported for WD mice treated with 1 ⁇ g of pT-LP1- hATP7B,C9 by hydrodynamic injection. Mice were bled at 20 weeks age, and six weeks post- DNA injection.
  • ATP7B KO mice were treated with 1 ⁇ g of pT-LP1-hATP7B,C9, while control groups of untreated heterozygous and KO mice were also analyzed.
  • Albumin (FIG.5A), Alkaline phosphatase (FIG.5B), total bilirubin (FIG.5C), total protein (FIG.5D), and glucose (FIG.5E) are presented.
  • Statistical significance was calculated with unpaired, parametric t-tests, * p ⁇ 0.05, ** p ⁇ 0.005, *** p ⁇ 0.0005, n.s. non-significant.
  • FIG. 6A-D shows hydrodynamic injection of 25 ⁇ g of pT-LP1-hATP7B,C9 into WD mice did not yield improvements in liver injury.
  • FIG. 6A the experimental plan is depicted: male and female WD mice were hydrodynamically injected with 25 ⁇ g of pT-LP1-hATP7B,C9 and 10 ⁇ g pCMV-hyPBase, the latter for integration. Mice were injected prior to WD liver phenotype development, and mice were harvested at 20 weeks for further analysis. The same female heterozygous and KO mice depicted in FIG. 2 are presented here as control groups for comparison. Serum chemistries for ALT (FIG. 6B), AST (FIG.
  • FIGS. 7A-C shows hydrodynamic injection of 25 ug of pT-LP1-hATP7B,C9 mediates high-level expression in mouse liver, but expression is lost over time.
  • FIG. 7A shows that hydrodynamic injection of 25 ⁇ g of pT-LP1-hATP7B,C9 and 10 ⁇ g pCMV-hyPBase mediates high-level expression and transfection efficiency in mice 4 days after transfection.
  • FIG.7C shows mice that are 12 weeks post-hydrodynamic injection of 25 ⁇ g of pT-LP1-hATP7B,C9 and 10 ⁇ g pCMV- hyPBase display were examined by immunofluorescence for ATP7B,C9 expression using the 1D4 antibody and anti-mouse FITC secondary. Mice harvested 4 days post-injection served as a control.
  • FIGS.8A-D shows hydrodynamic injection of 5 ug of pT-LP1-hATP7B,C9 into WD mice did not yield improvements in liver injury.
  • FIG.8A the experimental plan is depicted: male and female WD mice were hydrodynamically injected with 5 ⁇ g of pT-LP1-hATP7B,C9 and 5 ⁇ g pCMV-hyPBase, the latter for integration. Mice were injected prior to WD liver phenotype development, and mice were harvested at 20 weeks for further analysis. The same female heterozygous and KO mice depicted in FIG.2 are presented here as control groups for comparison. Serum chemistries for ALT (FIG. 8B), AST (FIG.
  • the present disclosure relates to compositions and methods for treating Wilson's disease. More particularly, the present disclosure relates to compositions and methods for treating Wilson's disease by gene therapy. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that non-viral modification of hepatocytes with ATP7B gene in Wilson’s Disease (WD) liver can lead to expansion of those hepatocytes through a proliferative advantage.
  • WD Wilson’s Disease
  • This phenomenon had not previously observed or reported in other gene therapy or cell therapy strategies. This feature was accomplished through stable expression of ATP7B in host hepatocytes, which can be accomplished through transposons or other integrating strategies. Selection was also facilitated through even dispersion of gene vector throughout liver tissue with hydrodynamic injection.
  • the disclosure further describes vectors that also yield expression in cholangiocytes in addition to hepatocytes, which are also important in Wilson’s Disease pathology but not targeted in other gene therapy strategies. Targeting cholangiocytes further increases export of copper into the bile. This is accomplished through the use of novel synthetic promoters that drive expression in both cell types.
  • Wilson disease is a monogenic liver disease that results in the buildup of toxic levels of copper in different tissues, primarily affecting the liver and brain (Cz ⁇ onkowska et al 2018).
  • WD is caused by various mutations in ATP7B, which codes for a copper transporting transmembrane protein. The WD-causing mutations in ATP7B disrupt protein stability, intracellular localization, and copper transporting function.
  • WD is a autosomal recessive disorder, and the commonly observed compound heterozygous mutations produce a broad spectrum of disease-onset and manifestations (Cz ⁇ onkowska et al 2018).
  • the liver disease can eventually progress to cirrhosis and liver failure, while the brain toxicity can result in neuropsychiatric symptoms.
  • Treatments for WD include penicillamine and trientine, which are copper chelating agents that facilitates copper removal from the body, reducing tissue damage.
  • penicillamine can have significant toxicities resulting in poor compliance among WD patients (Mase ⁇ bas et al 2019). Lack of compliance can lead to ongoing copper toxicities with patients continuing to progress in disease pathology. Given that WD is a single-gene disorder, it is an attractive target for gene therapy.
  • ATP7B is expressed in many different tissues, the replacement of ATP7B gene has primarily focused on the liver. Liver plays a central role in the whole-body copper homeostasis and ATP7B is essential for this liver function: ATP7B facilitates the delivery of copper to ceruloplasmin (the major copper containing protein in a serum) and exports excess copper into bile. Liver transplants are able to cure WD in patients developing liver failure, and recent clinical studies suggest that they may even improve neuropsychiatric symptoms (Poujois et al 2020). Liver transplants are, however, a risky procedure with numerous comorbidities and require the lifelong maintenance of immunosuppression to ensure graft survival.
  • ATP7B Molecular Genetics Wilson’s Disease (WD) manifestations range from mild hepatic inflammation and tremor to cirrhosis, fulminant liver failure, depression, and psychotic episodes. The exact cause of this phenotypic variability remains poorly understood. Over 600 WD-causing mutations have been identified in ATP7B and increasing number of those mutations has been characterized. These studies revealed a spectrum of biochemical and cellular effects, ranging from a complete loss of ATP7B expression and function to potentially milder effects on ATP7B trafficking or stability.
  • ATP7B adenosine triphosphatase, copper transporting, beta polypeptide
  • ATP7B adenosine triphosphatase, copper transporting, beta polypeptide
  • adeno-associated viruses as the delivery vehicle for ATP7B, significant improvements in liver enzyme function and reductions in copper in the liver and other tissues were achieved (Murillo et al 2016; Murillo et al 2019).
  • AAV adeno-associated viruses
  • a chief problem using AAV has been the size limitation of 4.8 kB of the vector; the ATP7B cDNA itself is ⁇ 4.4 kB leaving not enough room for the gene expression elements and AAV ITR’s.
  • Prior art studies have utilized truncated forms of ATP7B with several of the metal binding domains deleted (Leng et al 2019), but these truncated forms may have altered protein function and regulation compared to the full-length protein.
  • Non-viral approaches without size restriction could be utilized.
  • Hydrodynamic gene delivery can deliver naked plasmid DNA (pDNA) directly into hepatocytes in mice (Liu et al 1999) but has not previously been explored to deliver ATP7B for WD gene therapy.
  • the present disclosure provides methods and compositions for an effective gene therapy for WD.
  • the disclosure provides a technique of non-viral hydrodynamic delivery to deliver the full-length ATP7B cDNA into hepatocytes.
  • the disclosure provides a vector composition and method to endow hepatocytes expressing ATP7B with a proliferative advantage over non-transfected hepatocytes, which is optionally aided by using a transposon system to mediate integration into the hepatocyte genome.
  • the disclosure provides a method to achieve efficient ATP7B expression in the liver of human-sized large animals at clinically relevant, disease-modifying levels. No previous gene delivery studies have provided methods and compositions that achieved hATP7B gene delivery into large animals.
  • the present disclosure provides a method of efficient ATP7B expression from a non-viral vector that paves the way for clinical translation.
  • Wilson's disease represents a potential cure for Wilson's disease, which is caused by mutations of the ATP7B gene leading to decreased function of the protein in transporting copper.
  • the lack of normal ATP7B function leads to the buildup of copper in the liver, brain and other tissues, eventually leading to organ damage in these tissues and various clinical signs and symptoms.
  • Previous attempts at gene therapy for Wilson’s Disease have focused the delivery of ATP7B with adeno-associated virus (AAV) vectors.
  • AAV vectors have size limitations of around 4.7-8 kb in total. This size limitation makes it challenging to use the platform as an ATP7B delivery vehicle, given that ATP7B cDNA has a vector size of 4.4 kb for the protein coding sequence.
  • deletion variants have the same function long-term as full-length ATP7B.
  • microdystrophin genes for Duchenne s muscular dystrophy proved effective in mouse models after AAV gene therapy but have proven to be ineffective in actual human patients (https://www.evaluate.com/vantage/articles/news/trial-results/gene-therapy-trial-fails-rectify- sareptas-sorry-record).
  • the promoters utilized consist of small, liver-specific promoters driven by ubiquitous high-level expression and lack regulation of ATP7B in response to copper metabolism.
  • the prior art presents several limitations. Full-length ATP7B protein is not being utilized, promoter elements to do not feature native regulation of ATP7B expression in sensing copper status.
  • AAV-based platforms represent the disadvantages of immune responses against the capsid, preventing redosing of AAV and causing acute toxicity. Many patients are excluded from trials because of pre-existing antibodies against the AAV capsid. Post-administration, T cell responses develop against the capsid and can destroy transgene reduced cells.
  • the current disclosure describes a DNA vector for expressing ATP7B with several important improvements, which will be summarized as follows: The current disclosure envisions the delivery of a naked DNA molecule into hepatocytes of an individuals with Wilson's disease, with the DNA molecule entering into cell and eventually the nucleus of hepatocytes yielding expression.
  • the DNA molecule in most embodiments will be a circular DNA molecule, itself either a plasmid DNA molecule, or derived from a plasmid DNA molecule.
  • the DNA molecule maybe a linear DNA molecule with covalently closed ends having the bacterial sequences removed from the vector.
  • there are no distinct size limitations for the DNA vector encoding ATP7B although optimal forms of the DNA vector composition will preferably be as small as possible to increase delivery efficiency and/or yield from DNA manufacturing. This was demonstrated by delivering 8.4 kb size of plasmid DNA into the livers of mice and pigs by hydrodynamic gene delivery, respectively.
  • the promoter sequence is a crucial aspect to designing any gene therapy.
  • AAV gene therapy has significant size limitations, which has focused the AAV gene therapy field on small, short promoters that are expected to have high expression activity. Many smaller promoters are largely unregulated with respect to the gene being encoded, lacking the native gene-specific regulation of many genes to sense extracellular and intracellular conditions. For the purposes of ATP7B gene therapy, an unexpected and surprising result of approaches that leverages high-level, unregulated expression has been noted.
  • the first solution is that the method of gene therapy can be adjusted, wherein lower concentrations of plasmid DNA encoding ATP7B under the direction of a high level liver-specific promoter is performed. This would serve to titrate the DNA dose and subsequently the amount of ATP7B expression inside the cell.
  • ATP7B could be expressed from promoters that are regulated by the amount of copper and/or metals in the environment. Examples of these promoters include the native ATP7B promoter itself, as well as the metallothionein promoter.
  • hybrid promoters are contemplated such that the core promoter is a ubiquitous liver-specific promoter such as alpha-one antitrypsin (AAT) promoter, but the enhancer element that upregulates promoter expression will be based on a metal responsive element (MRE) found in the ATP7B or metallothionein promoters.
  • MRE metal responsive element
  • the native ATP7B promoter without any sequence alterations will be utilized up to 1500, 1200 or 1000 bp’s ahead of the native ATP7B will be utilized. Since the exact ATP7B promoter is undefined, and multiple different sequence lengths as listed could also be utilized.
  • a synthetic promoter with multiple repeats of MRE elements will be utilized to drive ATP7B expression.
  • a core region of the ATP7B promoter will be utilized, which encodes sequences containing only the positive regulatory elements of ATP7B promoter (-800 bp ahead of the native start codon of ATP7B gene) resulting in higher expression.
  • the negative regulatory regions of the ATP7B promoter will be utilized be kept in.
  • ATP7B promoters beyond using different sequence lengths of the native ATP7B promoter are envisioned. These primarily focus on the addition of enhancer elements to the ATP7B promoter to augment gene expression while maintaining regulation. While ATP7B promoters have the benefit of maintaining natural regulatory process to sense copper levels, the limitation is that the transcript level is relatively low compared to other liver-specific promoters used in gene therapy. This is particularly important when using episomal plasmid DNA vectors, which have generally lower expression of mRNA compared to the same gene on the host chromosome.
  • the ATP7B promoter is significantly weaker versus the native alpha-1 antitrypsin (AAT) promoter by comparison, which corresponds to the relative amounts of each proteins that are required for normal human physiology. Indeed, the ATP7B native promoter off an episomal DNA is only weakly active in mouse liver compared to the AAT promoter. To address these limitations, liver-specific enhancer elements such as APO HCR and ApoE enhancer are envisioned to be added to the 5’ end of the ATP7B promoter in certain embodiments of the disclosure.
  • AAT alpha-1 antitrypsin
  • enhancers derived from different viruses such as simian virus 40 and hepatitis B virus could also be added, which have general properties of increasing promoter strength, while maintaining the specificity of cell-type expression and regulation.
  • these enhancer elements include SV40 enhancer and HBV enhancer I and HBV enhancer II.
  • negative regulatory elements could be removed from the ATP7B promoter in the region -1200 to -800 and replaced with liver-specific or viral enhancers to enhance the expression ATP7B.
  • a liver specific core promoter could still be utilized, but the enhancer element could be derived from metal regulatory elements (MRE’s) that are governed by transcription factors, which sense the concentrations of different metals inside the cell, including copper.
  • MRE metal regulatory elements
  • a DNA sequence that includes multiple different copies of these metal regulatable elements (MRE’s) could be included 5’ to a core liver specific promoter such as alpha- 1 antitrypsin, in order to achieve effective enhancement of gene expression in response to the levels of metal inside the cell.
  • Alternative promoters beyond ATP7B can also be contemplated for the DNA vector.
  • Metallothionein is an important protein that helps sequester different metal ions in the body.
  • the metallothionein promoter also responds to increased levels of metal ions in order to facilitate more production of metallothionein protein to sequester the metal ions.
  • the metallothionein promoter is another alternative to regulate ATP7B expression inside hepatocytes.
  • Other elements of the DNA vector are also crucial for optimizing expression of the ATP7B gene, particularly in the situation where the promoter is relatively weak and is being regulated by copper status. In these situations, other elements can be utilized to counteract the relatively weaker expression from plasmid DNA in order to achieve sufficiently high ATP7B levels that are still regulated by copper levels.
  • the 5’ UTR will have an intron introduced into it, in order to increase mRNA export from the nucleus an ultimately expression in the cytoplasm.
  • These introns will be non-native to the 5 UTR of human ATP7B, as well as the metallothionein or alpha-1 antitrypsin promoters contemplated. In optimal embodiments, it would include miniature intronic elements from certain viral sequences such as SV40 or MVM.
  • the native ATP7B 5’ UTR will be utilized in the vector, such that introns will be introduced into the coding sequence of the ATP7B protein, where they otherwise do not naturally exist.
  • At least one intron will be introduced, although other embodiments will employ the use of two or more introns.
  • a 3’ UTR would be added downstream of the ATP7B coding sequence that would lead to the enhancement of expression.
  • the 3’ UTR in these embodiments which function in order to increase the half-life of mRNA inside the cell’s cytoplasm, as well as by enhancing translational potency of a given mRNA molecule. This would effectively increase ATP7B expression without interrupting its ultimate regulation from its promoter element.
  • Examples of 3’ UTR elements that can be used for this purpose include the human alpha and beta globin gene 3’ UTR regions.
  • a disadvantage of AAV vectors and plasmid DNA vectors is their episomal status.
  • sequences could be added to the DNA vector that would allow for replication during mitosis in cells. These sequences would be derived from scaffold matrix attachment regions (S/MAR) elements, which could be included in the DNA vector to facilitate replication. In some embodiments, S/MAR elements would optimally be placed 3’ region to or 3’ UTR of the ATP7B gene cassette.
  • S/MAR sequence on the vector will facilitate effective replication of the episomal DNA vector in the disclosure with hepatocyte mitosis, such that hepatocytes expressing ATP7B would be protected from copper toxicity, and thus the percentage of positive hepatocytes will increase within the liver over time.
  • Another element of the vector would be a DNA sequence directing polyadenylation of all the mRNA transcripts generated, as is customary in most expression vectors.
  • polyadenylation sequences that could be included in this vector include SV40 polyadenylation sequence, human growth hormone polyadenylation sequence, bovine growth hormone polyadenylation sequence, and rabbit beta-globin polyadenylation sequence.
  • the coding sequence, itself, of ATP7B is a key feature of the vector, which can improve the vector potency and resultant therapeutic activity. In optimal embodiments of the disclosure, the full length ATP7B gene will be utilized.
  • the coding sequence may be interrupted with additional introns in order to increase expression, but every native amino acid to ATP7B will be coded for.
  • a small c-terminal protein tag may be added in order to distinguish between endogenous mutant ATP7B and the vector delivered wild-type ATP7B transgene.
  • This C terminal tag in preferred embodiments should have no disruption of native ATP7B function and/or trafficking within the cell.
  • Examples of c-terminal tags that could be utilized include the C9-tag derived from the c-terminal 9 amino acids from the human rhodopsin protein, and the c-myc tag.
  • the exact DNA sequence of ATP7B may be the same as that encoded in the human genome. In other preferred embodiments, the DNA coding sequence is codon optimize to increase the levels of usage of common DNA codons in the human cells, such that the overall protein expression is increased.
  • the DNA sequence will be completely different from the native human ATP7B sequence, but the protein coded will be the same wild-type ATP7B gene.
  • the coding sequence of ATP7B preferentially uses codons that are utilized in the liver at high levels.
  • different small nucleotide polymorphisms will be incorporated into the coding sequence of ATP7B.
  • ATP7B is an enormous protein (1,465 amino acids), and as such that there exists no canonical truly wild-type sequence. Different SNP’s exist in the human population, and it is not obvious which of them to utilize and in which combinations.
  • SNP ATP7B activity in its copper transporting properties.
  • SNPs existing at K832 and R952 will be incorporated to increase the copper transport activity of ATP7B. This will lead to higher overall reduction in copper levels in spite of lower amounts of ATP7B protein.
  • Search use of hyperactive variants of ATP7B for use in gene therapy has not been previously reported in the literature. This strategy is analogous to the use of hyperactive human FIX variants in the treatment of hemophilia B.
  • the DNA vector is optimally delivered through hydrodynamic injection. Routes of hydrodynamic injection include vascular or biliary routes.
  • the DNA vector In either route, the DNA vector would be dissolved in a pharmaceutically acceptable solution, such as normal saline, phosphate buffered solution, lactate ringer’s solution, or dextrose solution. Optimal pressure will be obtained that creates pores in cell membranes in order to deliver the DNA vector inside cells.
  • the DNA vector could be encapsulated within a lipid particle or a lipid nanoparticle to facilitate DNA protection and cell uptake.
  • the compositions of the lipids in these particles may vary. In particular, hydrodynamic delivery through biliary routes offers higher efficiency of hepatocyte transfection compared to vascular routes, while also serving to allow for gene delivery into cholangiocytes.
  • ATP7B natively expresses in hepatocytes and cholangiocytes.
  • Other gene therapy approaches with AAV only serve to target hepatocytes currently. It is reasonable that also targeting expression into cholangiocytes would better allow for enhanced copper excretion, which naturally occurs through the biliary tract. Therefore, the current disclosure is unique in targeting both cell types for enhanced gene therapy for Wilson’s Disease.
  • a C-terminal tag encoding the last nine amino acids from the human rhodopsin protein (e.g., C9-tag; FIG.1A) was added to ATP7B.
  • the C9-tag may be located at the protein's C-terminus and has commonly been used to tag membrane proteins. However, the C9-tag may also be used to tag an epitope that is located intracellularly (Molday et al 2014).
  • the hATP7B,C9 gene was cloned into a plasmid DNA vector driven by a liver-specific promoter, with expression cassette located within piggyBac terminal repeats to mediate integration.
  • ATP7B,C9 was first transfected into YST cells, which lack native ATP7B expression. Immunofluorescent staining demonstrated that ATP7B,C9 can be efficiently detected within anti-C9 antibody, with the staining pattern was indistinguishable from ATP7B (FIG.1C). Importantly, this staining co-localized with the trans-golgi network (TGN) at basal copper levels, indicating proper ATP7B,C9 localization.
  • TGN trans-golgi network
  • ATP7B,C9 was then assessed to determine if it retained proper movement inside cells in response to copper. In high copper conditions it was observed that ATP7B,C9 moved out in speckled pattern beyond the TGN in the expected pattern for wildtype ATP7B (FIG.1C). In low copper conditions, ATP7B,C9 remained in the TGN as expected. Functional assays with ATP7B,C9 were then performed. Copper transport into the secretory pathway by ATP7B,C9 was monitored by evaluating copper loading into the copper- dependent enzyme tyrosinase (Roy et al 2020). Formation of eumelanin pigment by tyrosinase can be visually appreciated, indicating successful. copper transport.
  • tyrosinase tyrosinase
  • Example 2 Validation function of ATP7B after hydrodynamic injection in a mouse model of Wilson’s disease The techniques herein sought next to validate the function of ATP7B,C9 after non-viral delivery of pDNA into WD mice. WD mice with pathology already present were treated, which starts around 12 weeks age in C57BL6 mice. This would show that gene delivery a WD liver with injury was possible, important seen most WD patients already have liver phenotype at diagnosis.
  • the techniques herein chose to employ a low dose of pDNA, 1 ⁇ g, was employed to see the impact of fewer hepatocytes transfected, and whether these transfected cells would expand over time with integrated hATP7B,C9.
  • Mice were injected around 14 weeks ago, and evaluated at 20 weeks (FIG. 2A), a timepoint used for analysis in a previous study (Muchenditsi et al). After only 6 weeks of therapy, several markers in the Wilson's disease treated mice could be significantly reduced, including ALT, AST, and LDH (FIGS.2B-D). Other markers (alkaline phosphatase, total bilirubin) did not show any significant difference from the untreated group (FIG.5).
  • Hepatic copper levels were also reduced by 27% in treated WD mice, indicating successful copper transport function of ATP7B,C9 in vivo (FIG.2E).
  • the presence of ATP7B,C9 hepatocytes was observed by immunohistochemistry for the C9-tag in the mice harvested at 20 weeks, confirming their presence to mediate the copper reduction (FIG.3A).
  • Hepatocytes exhibited a range of staining intensities, likely reflecting different expression.
  • the percentage of ATP7B,C9 hepatocytes was calculated to be 4.65% area stained of hepatocytes in WD mice injected with 1 ⁇ g of plasmid DNA (FIG.3B).
  • Example 3 Biliary hydrodynamic injection of ATP7B plasmid in pigs
  • hydrodynamic injection of hATP7B,C9 pDNA into a human-sized animal model was tested.
  • the systemic pressure increase from vascular hydrodynamic tail vein injection in mice is not applicable to human patients.
  • the techniques herein have pioneered a strategy of hydrodynamic injection through the biliary system into pigs, which efficiently branches into all lobes and contacts all hepatocytes (Kumbhari et al 2018; Huang et al 2021).
  • ATP7B,C9 pDNA 10 mg was injected into the first pig.
  • pig liver was harvested at day 1 post-injection demonstrating no abnormalities or lesions after biliary hydrodynamic injection. DNA was extracted from all lobes, and PCR testing was able to correctly localize ATP7B DNA in all of liver lobes including proximal and distal locations in the lobe compared to the injection site (data not shown). Evaluating for protein expression by immunohistochemistry (IHC), ATP7B,C9 could be detected in all pig liver lobes. As shown in FIG.
  • IHC immunohistochemistry
  • ATP7B,C9 protein in pig hepatocytes was confirmed by immunofluorescence staining (FIG.4D).
  • ATP7B,C9 was observed to be correctly located in pig hepatocytes, with a pattern distinguishing from endogenous ATP7B in pigs, as well as ATP7B in mouse hepatocytes.
  • hATP7B can be expressed from a hydrodynamically delivered plasmid vector in mice and pigs for the first time.
  • Non-viral hydrodynamic gene delivery of pDNA has no defined size limit for the DNA vector, in comparison to AAV vectors previously employed.
  • hATP7B full-length hATP7B may be preferable to the use of truncated miniature ATP7B, which lack several metal binding domains, and have been used in AAV studies (Leng et al 2019).
  • hydrodynamic gene delivery of hATP7B DNA could occur in a diseased WD liver yielding functional protein in WD mice, reducing liver injury and decreasing hepatic copper content.
  • the level of hATP7B hepatocyte transfection (4.6%) was not enough to completely cure WD disease in mice, however. This is consistent with previous WD gene therapy studies, which suggested a level of 20% is necessary for cure in a WD mouse model (Murillo et al 2019).
  • the differences between the present study and the previous reports may be the higher expression mediated by non-viral gene delivery of ATP7B, yielding more competitive hepatocytes, as well as the even distribution of ATP7B cells across the liver tissue, as opposed to cell therapy efforts.
  • the techniques herein were able to achieve greater than 20% of hepatocytes expressing hATP7B after gene delivery into pigs.
  • a hepatocyte cell therapy in Long-Evans cinnamon rat model of WD suggested complete correction with 20% of ATP7B-positive hepatocytes (Irani et al 2001).
  • a recent gene therapy study suggested correction above 20% is sufficient to normalize phenotype, with significant improvement in copper reduction observed above 10% (Murillo et al 2019).
  • AAV can be dosed to transduce the majority of hepatocytes in mice, while HTVI is limited only transfected at most 20% of hepatocytes, and in many studies 5-10%.
  • ssDNA to dsDNA conversion among AAV genomes is inefficient, however, leading to lower levels of hepatocytes actually expressing hATP7B, while hydrodynamic injection delivers pDNA that is expressed within hours after injection. This may have resulted in lower net levels of ATP7B expressed across more hepatocytes for AAV approaches, contrasting with the present study.
  • the study is also limited by the length of time, one day, during which ATP7B expression was analyzed in pigs. While the focus was on defining the relative transfection efficiency, future studies will explore the duration of ATP7B expression in pigs.
  • the techniques herein demonstrate gene delivery of hATP7B DNA into a human-sized animal model for the first time. It was demonstrated that the plasmid DNA-mediated expression of ATP7B creates functional protein in both tissue and mouse models as well. Given that the techniques herein use clinically available equipment and an ERCP procedure in routine clinical practice today, the present disclosure indicates that the technique may be readily translated into the treatment of Wilson's disease patients.
  • the carboxy-terminus tag corresponding to the 9 terminal acid amino residues of the bovine rhodopsin gene was added by PCR cloning onto the hATP7B gene.
  • the hATP7B,C9 gene was inserted into the pT-LP1-hFIX vector developed by the Kumbhari lab, removing the hFIX gene via XbaI and BglII restriction sites. This vector was previous constructed through gene synthesis (Bio Basic).
  • the LP1 promoter is derived from a composite of the human apolipoprotein hepatic control region and the human alpha-1-antitrypsin (hAAT) gene promoter (Nathwani et al 2006), while the entire expression cassette is located between piggyBac transposon terminal repeats to facilitate integration (Wilson et al 2007).
  • the plasmid, pCMV-hyPBase was previously synthesized by the Kumbhari lab encoding a hyperactive piggyBac transposase to facilitate gene integration (Doherty et al 2007).
  • DNA was prepared for injection using QIAgen Plasmid Maxi prep kit for mouse injections and ZymoPURETM II Plasmid Gigaprep Kit (Zymo Research) for pig injections.
  • Menkes disease fibroblast (YST) cells lacking active ATP7A and ATP7B, were seeded in 8-well chamber slides at a density of 0.01x106 cells per well. The next day, cells were co- transfected with 100 ng each of either pTyr plasmid alone or with either wt ATP7B plasmid or D1027A GFP-ATP7B (inactive mutant). 20 h after transfection, expression of ATP7B was confirmed by GFP signal in DA samples.
  • YST cells were seeded at a density of 8x103 cells per well in 8-well chamber slides in complete media (CM: DMEM, 10% FBS, 1% penicillin/streptomycin). After 48 h, the cells were transfected with 200 ng of C9-ATP7B plasmid using Lipofectamine LTX and Plus reagent system.
  • the media was changed to either basal (CM with 9 mg/ml cycloheximide), high Cu (basal + 100 uM Cu), or low Cu (basal+ 25 uM TTM) media and incubated for 3 h at 37°C.
  • the cells were washed with PBS, fixed using 4% PFA for 15 min, permeabilized with 0.1% Triton X- 100 for 15 min, and blocked with 5% BSA for 40 min at RT. After washing with PBS, the cells were incubated with primary antibodies in PBS with 0.1% Triton X-100 (mouse 1D4 at 1:400 or rabbit anti-7B at 1:400, sheep TGN46 at 1:600) for 1 h at RT.
  • the cells were washed twice with PBST for 5 min and once with PBS for 5 min. The cells were then incubated with secondary antibodies in PBS with 0.1% Triton X-100 (anti-mouse Alexa488 at 1:400 or anti-rabbit Alexa 488 at 1:1000, anti-sheep Alexa 555 at 1:1000) for 1 h at RT, protected from light. The cells were washed twice with PBST for 5 min and once with PBS for 5 min, dried, mounted using Fluoromount-DAPI, and cured in the dark. The cells were imaged using LSM 800 confocal microscope with 63x oil lens. Mouse experiments All mouse studies were conducted under an approved protocol #MO17M385 by the Johns Hopkins IACUC committee.
  • mice were injected under established protocols for hydrodynamic tail vein injection (Liu et al 1999). Briefly, mice were warmed under heat lamp to induce vasodilation of their lateral tail veins. Using a 27 gauge needle, pDNA in saline solution corresponding to 10% of the body weight was injected into the mice within 5-7 seconds.
  • Serum was obtained by retro-orbital bleed from mice, and chemistries analyzed by the Johns Hopkins Phenotyping Core. Serum with noted hemolysis were excluded from analysis for AST and LDH. Mice were euthanized and perfused with saline before liver harvest and tissue analysis. Biliary hydrodynamic injection Pig experiments under the approval of the University of Maryland Baltimore IACUC #0720003. A detailed protocol for biliary hydrodynamic injection was previously described (Kumbhari et al 2018; Huang et al 2021). Briefly, all pigs were anesthetized for the procedure and monitored throughout for heart rate, blood pressure, and ventilation. An endoscope was advanced through the mouth and eventually into the small intestine.
  • a catheter was next advanced through the ampulla of Vater into the common bile duct. After further catheter advancement, the balloon on the catheter was subsequently inflated within the common hepatic duct. The catheter was connected to a power injector (Medrad Mark V Arterion), and the pigs were injected with pDNA dissolved in normal saline solution. Injection proceeded at parameters of 40 mL volume at 4 mL/second. Blood draws were collected before and after hydrodynamic injection into pigs to monitor for liver toxicity. Immunostaining For both mouse and pig studies, the use of a C9-tag was leveraged to distinguish delivered hATP7B from host mouse and pig ATP7B.
  • the 1D4 monoclonal antibody clone (mouse, Santa Cruz, Cat# 57432), which reacts with the 9 carboxy-terminal amino acids from the bovine rhodopsin protein (C9-tag), was used.
  • a mouse-on-mouse (MOM) protocol was used to reduce background staining in mouse liver.
  • MOM mouse-on-mouse
  • a polyclonal ATP7B antibody (ThermoFisher Scientific) was utilized with FITC labeled secondary antibody, while 1D4 antibody was utilized with the secondary antibody, goat anti-Mouse Alexa 647.
  • EGFP tags affect cellular localization of ATP7B mutants.
  • Cichon G Willnow T, Herwig S, Uckert W, Lenderr P, Schmidt HH, et al.
  • Non-physiological overexpression of the low density lipoprotein receptor (LDLr) gene in the liver induces pathological intracellular lipid and cholesterol storage.
  • LDLr low density lipoprotein receptor

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Abstract

L'invention concerne des procédés et une composition de traitement de la maladie de Wilson avec un vecteur d'ADN non viral. La composition est constituée de descriptions d'un gène ATP7B piloté par des éléments d'expression qui produisent une expression de haut niveau. L'administration de cette composition a permis d'obtenir un avantage prolifératif pour les cellules hépatiques hébergeant le vecteur dans un foie atteint de la maladie de la Wilson, de telle sorte que ces cellules augmentent en nombre dans le temps. Le vecteur d'ADN contient également des variants de séquence uniques d'ATP7B avec une activité accrue pour la thérapie génique. L'invention concerne également des conceptions de vecteurs qui permettent une réplication mitotique. L'invention concerne des procédés d'administration du vecteur d'ADN dans le foie par l'intermédiaire d'une distribution à nu d'injection hydrodynamique.
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